U.S. patent application number 11/131646 was filed with the patent office on 2005-11-10 for synthesis of acyclic nucleoside derivatives.
This patent application is currently assigned to Medivir AB. Invention is credited to Bellettini, John, Bhagavatula, Lakshmi, Chang, Sou-Jen, Curty, Cynthia B., Gates, Bradley D., Hannick, Steven M., Kolaczkowski, Lawrence, Lannoye, Greg, Leanna, M. Robert, Lukin, Kirill A., Morton, Howard, Narayanan, Bikshandarkoil A., Patel, Ramesh R., Plata, Daniel, Rasmussen, Michael, Riley, David A., Shelat, Bhadra, Singam, Pulla Reddy, Spitz, Tiffany, Tian, Zhenping, Tien, Jien-Heh J., Wayne, Greg, Yang, Cheng-Xi, Zhang, Weijiang.
Application Number | 20050250795 11/131646 |
Document ID | / |
Family ID | 22443606 |
Filed Date | 2005-11-10 |
United States Patent
Application |
20050250795 |
Kind Code |
A1 |
Leanna, M. Robert ; et
al. |
November 10, 2005 |
Synthesis of acyclic nucleoside derivatives
Abstract
Methods and novel intermediates for the preparation of acyclic
nucleoside derivatives of the formula: 1 where one of R.sub.1 and
R.sub.2 is an amino acid acyl group and the other of R.sub.1 and
R.sub.2 is a --C(O)C.sub.3-C.sub.21 saturated or monounsaturated,
optionally substituted alkyl and R.sub.3 is OH or H; or a
pharmaceutically acceptable salt thereof.
Inventors: |
Leanna, M. Robert;
(Grayslake, IL) ; Hannick, Steven M.; (Highland
Park, IL) ; Rasmussen, Michael; (Kenosha, WI)
; Tien, Jien-Heh J.; (Vernon Hills, IL) ;
Bhagavatula, Lakshmi; (Vernon Hills, IL) ; Singam,
Pulla Reddy; (Des Plaines, IL) ; Gates, Bradley
D.; (Mount Prospect, IL) ; Kolaczkowski,
Lawrence; (Gurnee, IL) ; Patel, Ramesh R.;
(Chicago, IL) ; Wayne, Greg; (Vernon Hills,
IL) ; Lannoye, Greg; (Wildwood, IL) ; Zhang,
Weijiang; (Grayslake, IL) ; Tian, Zhenping;
(Grayslake, IL) ; Lukin, Kirill A.; (Mundelein,
IL) ; Narayanan, Bikshandarkoil A.; (Mundelein,
IL) ; Riley, David A.; (Kenosha, WI) ; Morton,
Howard; (Gurnee, IL) ; Chang, Sou-Jen;
(Prairie View, IL) ; Curty, Cynthia B.; (Gurnee,
IL) ; Plata, Daniel; (Wadsworth, IL) ;
Bellettini, John; (Waukegan, IL) ; Shelat,
Bhadra; (Lake Forest, IL) ; Spitz, Tiffany;
(Highland Park, IL) ; Yang, Cheng-Xi; (Glenview,
IL) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
Medivir AB
Huddinge
SE
|
Family ID: |
22443606 |
Appl. No.: |
11/131646 |
Filed: |
May 18, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11131646 |
May 18, 2005 |
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10871751 |
Jun 17, 2004 |
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10871751 |
Jun 17, 2004 |
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10315580 |
Dec 9, 2002 |
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6878844 |
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10315580 |
Dec 9, 2002 |
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09692599 |
Oct 18, 2000 |
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6613936 |
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09692599 |
Oct 18, 2000 |
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09130214 |
Aug 6, 1998 |
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6184376 |
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09130214 |
Aug 6, 1998 |
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09020231 |
Feb 6, 1998 |
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60055153 |
Aug 8, 1997 |
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60037517 |
Feb 10, 1997 |
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Current U.S.
Class: |
514/263.38 ;
544/276 |
Current CPC
Class: |
C07D 473/00 20130101;
C07D 473/18 20130101; C07C 309/73 20130101; Y02P 20/55
20151101 |
Class at
Publication: |
514/263.38 ;
544/276 |
International
Class: |
C07D 473/12; A61K
031/522 |
Claims
1. A process for the preparation of a compound of the formula:
41wherein R.sub.10 is C.sub.3-C.sub.21 saturated or
monounsaturated, optionally substituted alkyl and R.sub.11 is
isopropyl or isobutyl, comprising a) deprotecting the acetal of the
compound of the formula: 42wherein R.sub.6 and R.sub.7 are
loweralkyl or benzyl or R.sub.6 and R.sub.7 taken together are
--CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2-- and R.sub.10 is as defined
above; b) reducing the aldehyde substituent of the product of step
a) to provide the alcohol of the formula: 43wherein R.sub.10 is as
defined above; and c) reacting the product of step b) with
P.sub.1NHCH(R.sub.11)COOH or an activated derivative thereof or
with P.sub.1NHCH(R.sub.11)C(O)--O--C(O)CH- (R.sub.11)NHP.sub.1
wherein R.sub.11 is as defined above and P.sub.1 is an N-protecting
group.
2. The process of claim 1 further comprising N-deprotecting the
product of step c).
3. The process of claim 1 wherein R.sub.6 and R.sub.7 are
--CH.sub.3 or --CH.sub.2CH.sub.3 or R.sub.6 and R.sub.7 taken
together are --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, R.sub.11 is C.sub.9-C.sub.19
alkyl and P.sub.1 is t-butyloxycarbonyl or benzyloxycarbonyl.
4. The process of claim 1 wherein R.sub.6 and R.sub.7 are
--CH.sub.2CH.sub.3, R.sub.10 is --(CH.sub.2).sub.16CH.sub.3 and
P.sub.1 is t-butyloxycarbonyl or benzyloxycarbonyl.
5. The process of claim 1 wherein the acetal is deprotected by
reaction with an acid or an acidic resin.
6. The process of claim 1 wherein the aldehyde substituent of the
product of step a) is reduced with borane t-butyl amine
complex.
7. A process for purifying a compound of the formula: 44wherein
R.sub.10 is C.sub.3-C.sub.21 saturated or monounsaturated,
optionally substituted alkyl comprising reacting it with a chiral
organic sulfonic acid.
8. The process of claim 7 wherein R.sub.10 is
--(CH.sub.2).sub.16CH.sub.3 and the sulfonic acid is
(S)-(+)-camphorsulfonic acid.
9. A process for the preparation of a compound of the formula:
45wherein R.sub.6 and R.sub.7 are loweralkyl or benzyl or R.sub.6
and R.sub.7 taken together are --CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2-- and R.sub.10 is
C.sub.3-C.sub.21 saturated or monounsaturated, optionally
substituted alkyl, comprising reacting a compound of the formula:
46wherein R.sub.6 and R.sub.7 are as defined above with
R.sub.10COOH or an activated derivative thereof.
10. The process of claim 9 wherein the activated derivative of
R.sub.10COOH is R.sub.10C(O)OS(O).sub.2R.sub.30 wherein R.sub.30 is
loweralkyl, phenyl or toluyl or R.sub.10C(O)OC(O)R.sub.10 or
R.sub.10C(O)OC(O)R.sub.10a wherein R.sub.10a is loweralkyl and
wherein R.sub.10 is --(CH.sub.2).sub.16CH.sub.3.
11. The process of claim 9 wherein the activated derivative of
R.sub.10COOH is
CH.sub.3(CH.sub.2).sub.16C(O)OC(O)C(CH.sub.3).sub.3.
12. The process of claim 11 wherein
CH.sub.3(CH.sub.2).sub.16C(O)OC(O)C(CH- .sub.3).sub.3 is prepared
in situ.
13. A process for the purification of a compound of the formula:
47wherein R.sub.4 and R.sub.5 are loweralkyl or benzyl and R.sub.6
and R.sub.7 are lower alkyl or benzyl or R.sub.6 and R.sub.7 taken
together are --CH.sub.2CH.sub.2-- or --CH.sub.2CH.sub.2CH.sub.2--
or --CH.sub.2CH.sub.2CH.sub.2CH.sub.2-- comprising reacting the
compound with dilute aqueous base.
14. A process for the preparation of a compound of the formula:
48wherein R.sub.6 and R.sub.7 are lower alkyl or benzyl or R.sub.6
and R.sub.7 taken together are --CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2-- comprising reacting a compound
of the formula: 49wherein R.sub.6 and R.sub.7 are as defined above
and R.sub.8 is C.sub.1-C.sub.21 saturated or monounsaturated,
optionally substituted alkyl with an inorganic base.
15. The process of claim 14 wherein the inorganic base is KOH or
NaOH.
16. A process for the preparation of a compound of the formula:
50wherein R.sub.6 and R.sub.7 are loweralkyl or benzyl or R.sub.6
and R.sub.7 taken together are --CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, and R.sub.8 is
C.sub.1-C.sub.21 saturated or monounsaturated, optionally
substituted alkyl, comprising reacting a compound of the formula:
51wherein R.sub.6 and R.sub.7 are as defined above with
CH.sub.2.dbd.CH--OC(O)R.sub.8 wherein R.sub.8 is as defined above,
in the presence of a lipase.
17. The process of claim 16 wherein R.sub.6 and R.sub.7 are
--CH.sub.3 or --CH.sub.2CH.sub.3 or R.sub.6 and R.sub.7 taken
together are --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, and R.sub.8 is
--(CH.sub.2).sub.16CH.sub.3.
18. The process of claim 16 wherein R.sub.6 and R.sub.7 are
--CH.sub.2CH.sub.3 and R.sub.8 is --(CH.sub.2).sub.16CH.sub.3.
19. A process for the preparation of a compound of the formula:
52wherein R.sub.6 and R.sub.7 are loweralkyl or benzyl or R.sub.6
and R.sub.7 taken together are --CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, and R.sub.8 is
C.sub.1-C.sub.21 saturated or monounsaturated, optionally
substituted alkyl and X.sub.2 is a leaving group, comprising a)
reacting a compound of the formula: 53wherein R.sub.6 and R.sub.7
are as defined above with CH.sub.2.dbd.CH--OC(O)R.sub.8 wherein
R.sub.8 is as defined above, in the presence of a lipase to provide
an ester of the formula: 54wherein R.sub.6, R.sub.7 and R.sub.8 are
as defined above; and b) converting the hydroxyl group of the
product of step a) to a leaving group.
20. The process of claim 19 wherein R.sub.6 and R.sub.7 are
--CH.sub.3 or --CH.sub.2CH.sub.3 or R.sub.6 and R.sub.7 taken
together are --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, and R.sub.8 is
--(CH.sub.2).sub.16CH.sub.3 and X.sub.2 is a halogen or a sulfonate
leaving group.
21. The process of claim 19 wherein R.sub.6 and R.sub.7 are
--CH.sub.2CH.sub.3 and R.sub.8 is --(CH.sub.2).sub.16CH.sub.3 and
X.sub.2 is tosylate.
22. A compound of the formula: 55wherein R.sub.6 and R.sub.7 are
loweralkyl or benzyl or R.sub.6 and R.sub.7 taken together are
--CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, R.sub.8 is C.sub.1-C.sub.21
saturated or monounsaturated, optionally substituted alkyl and
R.sub.9 is an alcohol protecting group.
23. The compound of claim 22 wherein R.sub.6 and R.sub.7 are
--CH.sub.3 or --CH.sub.2CH.sub.3 or R.sub.6 and R.sub.7 taken
together are --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2-- and R.sub.8 is CH.sub.3.
24. The compound of claim 22 wherein R.sub.6 and R.sub.7 are
--CH.sub.2CH.sub.3 and R.sub.8 is CH.sub.3.
25. The compound of claim 22 wherein R.sub.6 and R.sub.7 are
--CH.sub.3 or --CH.sub.2CH.sub.3 or R.sub.6 and R.sub.7 taken
together are --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2-- and R.sub.8 is
--(CH.sub.2).sub.16CH.sub.3.
26. The compound of claim 22 wherein R.sub.6 and R.sub.7 are
--CH.sub.2CH.sub.3 and R.sub.8 is --(CH.sub.2).sub.16CH.sub.3.
27. A compound of the formula: 56wherein R.sub.6 and R.sub.7 are
loweralkyl or benzyl or R.sub.6 and R.sub.7 taken together are
--CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2-- and R.sub.9 is H or an alcohol
protecting group.
28. The compound of claim 27 wherein R.sub.6 and R.sub.7 are
--CH.sub.3 or --CH.sub.2CH.sub.3 or R.sub.6 and R.sub.7 taken
together are --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2-- and R.sub.9 is benzyl.
29. The compound of claim 28 wherein R.sub.6 and R.sub.7 are
--CH.sub.2CH.sub.3 and R.sub.9 is benzyl.
30. A compound of the formula: 57wherein R.sub.6 and R.sub.7 are
loweralkyl or benzyl or R.sub.6 and R.sub.7 taken together are
--CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2-- and R.sub.10 is
C.sub.3-C.sub.21 saturated or monounsaturated, optionally
substituted alkyl.
31. The compound of claim 30 wherein R.sub.6 and R.sub.7 are
--CH.sub.3 or --CH.sub.2CH.sub.3 or R.sub.6 and R.sub.7 taken
together are --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2-- and R.sub.10 is
--(CH.sub.2).sub.16CH.sub.3.
32. The compound of claim 30 wherein R.sub.6 and R.sub.7 are
--CH.sub.2CH.sub.3 and R.sub.10 is --(CH.sub.2).sub.16CH.sub.3.
33. A compound of the formula: 58wherein R.sub.6 and R.sub.7 are
loweralkyl or benzyl or R.sub.6 and R.sub.7 taken together are
--CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2-- and R.sub.8 is
C.sub.1-C.sub.21 saturated or monounsaturated, optionally
substituted alkyl.
34. The compound of claim 33 wherein R.sub.6 and R.sub.7 are
--CH.sub.3 or --CH.sub.2CH.sub.3 or R.sub.6 and R.sub.7 taken
together are --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2-- and R.sub.8 is
--(CH.sub.2).sub.16CH.sub.3.
35. The compound of claim 33 wherein R.sub.6 and R.sub.7 are
--CH.sub.2CH.sub.3 and R.sub.8 is --(CH.sub.2).sub.16CH.sub.3.
36. A compound of the formula: 59wherein R.sub.6 and R.sub.7 are
loweralkyl or benzyl or R.sub.6 and R.sub.7 taken together are
--CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2-- and R.sub.8 is
C.sub.1-C.sub.21 saturated or monounsaturated, optionally
substituted alkyl and X.sub.2 is a leaving group.
37. The compound of claim 36 wherein R.sub.6 and R.sub.7 are
--CH.sub.3 or --CH.sub.2CH.sub.3 or R.sub.6 and R.sub.7 taken
together are --CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2-- and R.sub.8 is
--(CH.sub.2).sub.16CH.sub.3 and X.sub.2 is a halogen or a sulfonate
leaving group.
38. The compound of claim 36 wherein R.sub.6 and R.sub.7 are
--CH.sub.2CH.sub.3 and R.sub.8 is --(CH.sub.2).sub.16CH.sub.3 and
X.sub.2 is tosylate.
39. A process for the preparation of a compound of the formula:
60wherein R.sub.10 is C.sub.3-C.sub.21 saturated or
monounsaturated, optionally substituted alkyl and R.sub.11 is
isopropyl or isobutyl, comprising a) deprotecting the ether
substituent of the compound of the formula: 61wherein R.sub.12 is
--CH(Ph).sub.2, --C(Ph).sub.3 or --Si(t-Bu)(CH.sub.3).sub.2 wherein
Ph is phenyl and R.sub.10 is as defined above to provide the
alcohol of the formula: 62wherein R.sub.10 is as defined above; and
b) reacting the product of step a) with P.sub.1NHCH(R.sub.11)COOH
or an activated derivative thereof or with
P.sub.1NHCH(R.sub.11)C(O)--O--C(O)CH(R.sub.11)NHP.sub.1 wherein
R.sub.11 is as defined above and P.sub.1 is an N-protecting
group.
40. The process of claim 39 further comprising N-deprotecting the
product of step b).
41. The process of claim 39 wherein R.sub.12 is as defined therein,
R.sub.10 is C.sub.9-C.sub.19 alkyl and P.sub.1 is
t-butyloxycarbonyl or benzyloxycarbonyl.
42. The process of claim 39 wherein R.sub.12 is as defined therein,
R.sub.10 is --(CH.sub.2).sub.16CH.sub.3 and P.sub.1 is
t-butyloxycarbonyl or benzyloxycarbonyl.
43. A process for the preparation of a compound of the formula:
63wherein R.sub.8 is C.sub.1-C.sub.21 saturated or monounsaturated,
optionally substituted alkyl and R.sub.12 is --CH(Ph).sub.2,
--C(Ph).sub.3 or --Si(t-Bu)(CH.sub.3).sub.2 wherein Ph is phenyl,
comprising reacting a compound of the formula: 64wherein R.sub.12
is as defined above with CH.sub.2.dbd.CH--OC(O)R.sub.8 wherein
R.sub.8 is as defined above, in the presence of a lipase.
44. The process of claim 43 wherein R.sub.12 is as defined therein
and R.sub.8 is --(CH.sub.2).sub.16CH.sub.3.
45. A process for the preparation of a compound of the formula:
65wherein R.sub.8 is C.sub.1-C.sub.21 saturated or monounsaturated,
optionally substituted alkyl, R.sub.12 is --CH(Ph).sub.2,
--C(Ph).sub.3 or --Si(t-Bu)(CH.sub.3).sub.2 wherein Ph is phenyl,
and X.sub.2 is a leaving group, comprising a) reacting a compound
of the formula: 66wherein R.sub.12 is as defined above with
CH.sub.2.dbd.CH--OC(O)R.sub.8 wherein R.sub.8 is as defined above,
in the presence of a lipase to provide an ester of the formula:
67wherein R.sub.8 and R.sub.12 are as defined above; and b)
converting the hydroxyl group of the product of step a) to a
leaving group.
46. The process of claim 45 wherein R.sub.8 is
--(CH.sub.2).sub.16CH.sub.3 and R.sub.12 is as defined therein and
X.sub.2 is a halogen or a sulfonate leaving group.
47. The process of claim 45 wherein R.sub.8 is
--(CH.sub.2).sub.16CH.sub.3 and R.sub.12 is as defined therein and
X.sub.2 is tosylate.
48. A compound of the formula: 68wherein R.sub.8 is
C.sub.1-C.sub.21 saturated or monounsaturated, optionally
substituted alkyl and R.sub.12 is --CH(Ph).sub.2, --C(Ph).sub.3 or
--Si(t-Bu)(CH.sub.3).sub.2 wherein Ph is phenyl.
49. The compound of claim 48 wherein R.sub.8 is CH.sub.3 and
R.sub.12 is as defined therein.
50. The compound of claim 48 wherein R.sub.8 is
--(CH.sub.2).sub.16CH.sub.- 3 and R.sub.12 is as defined
therein.
51. A compound of the formula: 69wherein R.sub.9 is H or an alcohol
protecting group and R.sub.12 is --CH(Ph).sub.2, --C(Ph).sub.3 or
--Si(t-Bu)(CH.sub.3).sub.2 wherein Ph is phenyl.
52. The compound of claim 51 wherein R.sub.9 is benzyl and R.sub.12
is as defined therein.
53. A compound of the formula: 70wherein R.sub.10 is
C.sub.3-C.sub.21 saturated or monounsaturated, optionally
substituted alkyl and R.sub.12 is --CH(Ph).sub.2, --C(Ph).sub.3 or
--Si(t-Bu)(CH.sub.3).sub.2 wherein Ph is phenyl.
54. The compound of claim 53 wherein R.sub.10 is
--(CH.sub.2).sub.16CH.sub- .3 and R.sub.12 is as defined
therein.
55. A compound of the formula: 71wherein R.sub.8 is
C.sub.1C-.sub.21 saturated or monounsaturated, optionally
substituted alkyl and R.sub.12 is --CH(Ph).sub.2, --C(Ph).sub.3 or
--Si(t-Bu)(CH.sub.3).sub.2 wherein Ph is phenyl.
56. The compound of claim 55 wherein R.sub.8 is CH.sub.3 and
R.sub.12 is as defined therein.
57. The compound of claim 55 wherein R.sub.8 is
--(CH.sub.2).sub.16CH.sub.- 3 and R.sub.12 is as defined
therein.
58. A compound of the formula: 72wherein R.sub.8 is
C.sub.1-C.sub.21 saturated or monounsaturated, optionally
substituted alkyl, R.sub.12 is --CH(Ph).sub.2, --C(Ph).sub.3 or
--Si(t-Bu)(CH.sub.3).sub.2 wherein Ph is phenyl and X.sub.2 is a
leaving group.
59. The compound of claim 58 wherein R.sub.8 is CH.sub.3, R.sub.12
is defined therein and X.sub.2 is a halogen or a sulfonate leaving
group.
60. The compound of claim 58 wherein R.sub.8 is CH.sub.3, R.sub.12
is as defined therein and X.sub.2 is tosylate.
61. The compound of claim 58 wherein R.sub.8 is
--(CH.sub.2).sub.16CH.sub.- 3, R.sub.12 is as defined therein and
X.sub.2 is a halogen or a sulfonate leaving group.
62. The compound of claim 58 wherein R.sub.8 is
--(CH.sub.2).sub.16CH.sub.- 3, R.sub.12 is as defined therein and
X.sub.2 is tosylate.
63. A method for the preparation of a compound of the formula I
73where a) R.sub.1 is --C(O)CH(CH(CH.sub.3).sub.2)NH.sub.2 or
--C(O)CH(CH(CH.sub.3)CH.sub.2CH.sub.3)NH.sub.2 and R.sub.2 is
--C(O)C.sub.3-C.sub.21 saturated or monounsaturated, optionally
substituted alkyl; or b) R.sub.1 is --C(O)C.sub.3-C.sub.21
saturated or monounsaturated, optionally substituted alkyl and
R.sub.2 is --C(O)CH(CH(CH.sub.3).sub.2)NH.sub.2 or
--C(O)CH(CH(CH.sub.3)CH.sub.2CH.s- ub.3)NH.sub.2; and R.sub.3 is OH
or H; the method comprising a) converting Q.sub.2 in a compound of
the formula 74where Q.sub.1 is a first protecting group, Q.sub.2 is
H, Q.sub.3 is optionally protected OH or a leaving group, and
Q.sub.4 is H or OQ.sub.1, to a second protecting group or a
--C(O)C.sub.3-C.sub.21 saturated or monounsaturated optionally
substituted alkyl derivative; b) converting Q.sub.3 to an N-9
guanine derivative; c) replacing said second protecting group, if
present, with a --C(O)CH(CH(CH.sub.3).sub.2)NH.sub.2 derivative, a
--C(O)CH(CH(CH.sub.3)CH.sub.2CH.sub.3)NH.sub.2 derivative or a
--C(O)C.sub.3-C.sub.21 saturated or monounsaturated optionally
substituted alkyl derivative; d) converting, if necessary, Q.sub.4
to hydrogen; and e) converting Q.sub.1 to a
--C(O)CH(CH(CH.sub.3).sub.2)NH.s- ub.2 derivative, a
--C(O)CH(CH(CH.sub.3)CH.sub.2CH.sub.3)NH.sub.2 derivative or a
--C(O)C.sub.3-C.sub.21 saturated or monounsaturated optionally
substituted alkyl derivative.
64. A process for the preparation of a compound of the formula:
75wherein R.sub.10 is C.sub.3-C.sub.21 saturated or
monounsaturated, optionally substituted alkyl and R.sub.11 is
isopropyl or isobutyl, comprising reacting a compound of the
formula: 76wherein R.sub.9 is an alcohol protecting group with a
compound of the formula: 77wherein X.sub.2 is a halogen or
sulfonate leaving group and P.sub.1 is an N-protecting group and
R.sub.10 and R.sub.11 are as defined above.
65. The process of claim 64 wherein R.sub.10 is
--(CH.sub.2).sub.16CH.sub.- 3 and X.sub.2 is
p-toluenesulfonyloxy.
66. The process of 64 wherein R.sub.9 is benzyl.
67. The process of claim 64 further comprising a base.
68. The process of claim 67 wherein the base is potassium
carbonate, LiH, NaH, KH, NaOH, KOH, lithium diisopropylamide,
LiN(Si(CH.sub.3).sub.3).sub- .2 or a sterically bulky amine
base.
69. The process of claim 68 wherein the sterically bulky amine base
is 1,8-diazabicyclo[5.4.0]undec-7-ene,
1,4-diazabicyclo[2.2.2]-octane, 1,5-diazabicyclo[4.3.0]non-5-ene,
tetramethylguanidine, N,N-diisopropylethylamine or a sterically
bulky phosphazine base.
70. A compound of the formula: 78wherein X.sub.2 is a halogen or
sulfonate leaving group, P.sub.1 is an N-protecting group, R.sub.10
is C.sub.3-C.sub.21 saturated or monounsaturated, optionally
substituted alkyl and R.sub.11 is isopropyl or isobutyl.
71. The compound of claim 70 wherein X.sub.2 is
p-toluenesulfonyloxy and R.sub.10 is
--(CH.sub.2).sub.16CH.sub.3.
72. The compound of claim 70 wherein X.sub.2 is
p-toluenesulfonyloxy, R.sub.10 is --(CH.sub.2).sub.16CH.sub.3,
R.sub.11 is isopropyl and P.sub.1 is benzyloxycarbonyl,
t-butyloxycarbonyl or allyloxycarbonyl.
73. A compound of the formula: 79wherein X.sub.2 is a halogen or
sulfonate leaving group and R.sub.10 is C.sub.3-C.sub.21 saturated
or monounsaturated, optionally substituted alkyl.
74. The compound of claim 73 wherein X.sub.2 is
p-toluenesulfonyloxy and R.sub.10 is
--(CH.sub.2).sub.16CH.sub.3.
75. A process for preparing a compound of the formula: 80wherein
R.sub.10 is C.sub.3-C.sub.21 saturated or monounsaturated,
optionally substituted alkyl comprising: a) reacting a compound of
the formula: 81wherein wherein R.sub.6 and R.sub.7 are loweralkyl
or benzyl or R.sub.6 and R.sub.7 taken together are
--CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub- .2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2-- with R.sub.1OCOOH or an
activated derivative thereof wherein R.sub.10 is C.sub.3-C.sub.21
saturated or monounsaturated, optionally substituted alkyl to
provide a compound of the formula: 82wherein R.sub.6, R.sub.7 and
R.sub.10 are defined as above, b) deprotecting the acetal of the
product of step a) and c) reducing the aldehyde substituent of the
product of step b).
76. The process of claim 75 wherein the products of steps a) and b)
are not isolated.
77. The process of claim 75 wherein R.sub.6 and R.sub.7 are
--CH.sub.2CH.sub.3 and R.sub.10 is --(CH.sub.2).sub.16CH.sub.3.
78. The process of claim 75 wherein the activated derivative of
R.sub.10COOH is
CH.sub.3(CH.sub.2).sub.16C(O)OC(O)C(CH.sub.3).sub.3.
79. The process of claim 75 wherein the acetal is deprotected with
triflic acid and the aldehyde substituent of the product of step b)
is reduced with borane t-butyl amine complex.
80. A process for preparing a compound of the formula: 83wherein
X.sub.2 is a halogen or sulfonate leaving group, P.sub.1 is an
N-protecting group, R.sub.10 is C.sub.3-C.sub.21 saturated or
monounsaturated, optionally substituted alkyl and R.sub.11 is
isopropyl or isobutyl comprising a) reducing to an alcohol the
aldehyde substituent of a compound of the formula: 84wherein
X.sub.2 and R.sub.1, are as defined above and b) reacting the
product of step a) with P.sub.1NHCH(R.sub.11)CO- OH or an activated
derivative thereof or with P.sub.1NHCH(R.sub.11)C(O)--O-
--C(O)CH(R.sub.11)NHP.sub.1 wherein R.sub.11 and P.sub.1 are as
defined above.
81. The process of claim 80 wherein the product of step a) is not
isolated
82. The process of claim 80 wherein X.sub.2 is p-toluenesulfonyloxy
and R.sub.10 is --(CH.sub.2).sub.16CH.sub.3.
83. The process of claim 80 wherein X.sub.2 is
p-toluene-sulfonyloxy, R.sub.10 is --(CH.sub.2).sub.16CH.sub.3,
R.sub.11 is isopropyl and P.sub.1 is benzyloxycarbonyl,
t-butyloxycarbonyl or allyloxycarbonyl.
84. A compound of the formula: 85wherein R.sub.9 is an alcohol
protecting group, R.sub.10 is C.sub.3-C.sub.21 saturated or
monounsaturated, optionally substituted alkyl, R.sub.11 is
isopropyl or isobutyl and P.sub.1 is an N-protecting group.
85. The compound of claim 84 wherein R.sub.10 is
CH.sub.3(CH.sub.2).sub.16- --, R.sub.11 is isopropyl and P.sub.1 is
benzyloxycarbonyl, t-butyloxycarbonyl or allyloxycarbonyl.
86. The compound of claim 84 wherein R.sub.9 is benzyl, R.sub.10 is
CH.sub.3(CH.sub.2).sub.16--, R.sub.11 is isopropyl and P.sub.1 is
benzyloxycarbonyl, t-butyloxycarbonyl, allyloxycarbonyl or
trichloroethylcarbonyl.
87. A process for the preparation of a compound of the formula:
86wherein R.sub.10 is C.sub.3-C.sub.21 saturated or
monounsaturated, optionally substituted alkyl and R.sub.11 is
isopropyl or isobutyl, comprising hydrolysis of a compound of the
formula: 87wherein R.sub.10 is C.sub.3-C.sub.21 saturated or
monounsaturated, optionally substituted alkyl, R.sub.11 is
isopropyl or isobutyl and P.sub.1 is an N-protecting group.
88. A process for the preparation of a compound of the formula:
88wherein R.sub.6 and R.sub.7 are loweralkyl or benzyl or R.sub.6
and R.sub.7 taken together are --CH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, R.sub.8 is C.sub.1-C.sub.21
saturated or monounsaturated, optionally substituted alkyl and
R.sub.9 is an alcohol protecting group comprising reacting a
compound of the formula: 89wherein R.sub.9 is an alcohol protecting
group with a compound of the formula: 90wherein R.sub.6, R.sub.7,
R.sub.8 and R.sub.9 are as defined above and X.sub.2 is a leaving
group.
89. The process of claim 88 wherein R.sub.8 is
--(CH.sub.2).sub.16CH.sub.3 or --CH.sub.3 and X.sub.2 is
p-toluenesulfonyloxy.
90. The process of 88 wherein R.sub.9 is benzyl.
91. The process of claim 88 further comprising a base.
92. The process of claim 91 wherein the base is potassium
carbonate, LiH, NaH, KH, NaOH, KOH, lithium diisopropylamide,
LiN(Si(CH.sub.3).sub.3).sub- .2 or a sterically bulky amine
base.
93. The process of claim 92 wherein the sterically bulky amine base
is 1,8-diazabicyclo[5.4.0]undec-7-ene,
1,4-diazabicyclo[2.2.2]-octane, 1,5-diazabicyclo[4.3.0]non-5-ene,
tetramethylguanidine, N,N-diisopropylethylamine or a sterically
bulky phosphazine base.
94. A compound of the formula: 91wherein R.sub.6 and R.sub.7 are
loweralkyl or benzyl or R.sub.6 and R.sub.7 taken together are
--CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--, R.sub.8 is C.sub.1-C.sub.21
saturated or monounsaturated, optionally substituted alkyl,
R.sub.25 is hydrogen or --C(O)NR.sub.27R.sub.28 wherein R.sub.27
and R.sub.28 are independently selected from loweralkyl, phenyl and
benzyl or R.sub.27 and R.sub.28, taken together with the nitrogen
to which they are attached, form a pyrrolidinyl group or a
piperidinyl group and R.sub.26 is loweralkyl, phenyl or benzyl.
95. The compound of claim 94 wherein R.sub.8 is
--(CH.sub.2).sub.16CH.sub.- 3 or --CH.sub.3, R.sub.25 is hydrogen
and R.sub.26 is --CH.sub.3.
96. The compound of claim 94 wherein R.sub.8 is
--(CH.sub.2).sub.16CH.sub.- 3 or --CH.sub.3, R.sub.25 is
--C(O)N(phenyl).sub.2 and R.sub.26 is --CH.sub.3.
97. A process for the preparation of a compound of the formula:
92wherein R.sub.10 is C.sub.3-C.sub.21 saturated, optionally
substituted alkyl and R.sub.11 is isopropyl or isobutyl, comprising
hydrogenation of a compound of the formula: 93wherein R.sub.10 and
R.sub.11 are as defined above and P.sub.1 is benzyloxycarbonyl with
a hydrogenation catalyst selected from Pd/BaSO.sub.4 and
Pd/BaCO.sub.3.
Description
[0001] This application is a divisional of co-pending application
Ser. No. 10/871,751, filed on Jun. 17, 2004, which is a divisional
of application Ser. No. 10/315,580, filed on Dec. 9, 2002, which is
a divisional of application Ser. No. 09/692,599, filed on Oct. 18,
2000, which is a divisional of application Ser. No. 09/130,214,
filed in the United States on Aug. 6, 1998, which is a
continuation-in-part of 09/020,231, filed in the United States on
Feb. 6, 1998, the entire contents of which are hereby incorporated
by reference and for which priority is claimed under 35 U.S.C.
.sctn. 120; and this application claims priority of provisional
Application No. 60/055,153, filed in the United States on Aug. 8,
1997 and also claims priority of provisional Application No.
60/037,517, filed in the United States on Feb. 10, 1997 under 35
U.S.C. .sctn. 119(e), the entire contents of which are hereby
incorporated by reference.
TECHNICAL FIELD
[0002] This invention relates to the field of antivirals and in
particular to derivatives of acyclic nucleosides useful against
herpes and retroviral infections and methods for their manufacture
and novel intermediates.
BACKGROUND OF THE INVENTION
[0003] The practical utility of many acyclic nucleosides is limited
by their relatively modest pharmacokinetics. A number of prodrug
approaches have been explored in an effort to improve the
bioavailability of acyclic nucleosides in general. One of these
approaches involves the preparation of ester derivatives,
particularly aliphatic esters, of one or more of the hydroxy groups
on the acyclic side chain.
[0004] European patent EP 165 289 describes the promising
antiherpes agent 9-[4-hydroxy-(2-hydroxymethyl)butyl]guanine,
otherwise known as H2G. European patent EP 186 640 discloses
6-deoxy H2G. European patent EP 343 133 discloses that these
compounds, particularly the R-(-) enantiomer, are additionally
active against retroviral infections such as HIV. Various
derivatives of H2G, such as phosphonates, aliphatic esters (for
example, the diacetate and the dipropionate) and ethers of the
hydroxy groups on the acyclic side chain are disclosed in EP 343
133. This patent also discloses methods for the preparation of
these derivatives comprising the condensation of the acyclic side
chain to the N-9 position of a typically 6-halogenated purine
moiety or, alternatively, the imidazole ring closure of a
pyrimidine or furazano-[3,4-d]-pyrimidine moeity or the pyrimidine
ring closure of an imidazole moiety, where the acyclic side chain
is already present in the precursor pyrimidine or imidazole moiety,
respectively. In the broadest description of each of these methods
the acyclic side chain is pre-derivatised but individual examples
also show a one-step diacylation of H2G with acetic or proprionic
anhydride and DMF.
[0005] Harnden, et al., J. Med. Chem. 32, 1738 (1989) investigated
a number of short chain aliphatic esters of the acyclic nucleoside
9-[4-hydroxy-(3-hydroxymethyl)butyl]guanine, otherwise known as
penciclovir, and its 6-deoxy analog. Famciclovir, a marketed
antiviral agent, is the diacetyl derivative of 6-deoxy
penciclovir.
[0006] Benjamin, et al., Pharm. Res. 4 No. 2, 120 (1987) discloses
short chain aliphatic esters of
9-[(1,3-dihydroxy-2-propoxy)-methyl]guanine, otherwise known as
ganciclovir. The dipropionate ester is disclosed to be the
preferred ester.
[0007] Lake-Bakaar, et al., discloses in Antimicrob. Agents
Chemother. 33 No. 1,110-112 (1989) diacetate and dipropionate
derivatives of H2G and monoacetate and diacetate derivatives of
6-deoxy H2G. The diacetate and dipropionate derivatives of H2G are
reported to result in only modest improvements in bioavailability
relative to H2G.
[0008] International patent application WO94/24134, published Oct.
27, 1994, discloses aliphatic ester prodrugs of the 6-deoxy N-7
analog of ganciclovir, including the di-pivaloyl, di-valeroyl,
mono-valeroyl, mono-oleoyl and mono-stearoyl esters.
[0009] International patent application WO93/07163, published Apr.
15, 1993 and International patent application WO94/22887, published
Oct. 13, 1994, both disclose mono-ester derivatives of nucleoside
analogs derived from mono-unsaturated C18 or C20 fatty acids. U.S.
Pat. No. 5,216,142, issued Jun. 1, 1993, also discloses long chain
fatty acid mono-ester derivatives of nucleoside analogs.
[0010] A second approach to providing prodrugs of acyclic
nucleosides involves the preparation of amino acid esters of one or
more of the hydroxy groups on the acyclic side chain. European
patent EP 99 493 discloses generally amino acid esters of acyclovir
and European patent application EP 308 065, published Mar. 22,
1989, discloses the valine and isoleucine esters of acyclovir.
[0011] European patent application EP 375 329, published Jun. 27,
1990, discloses amino acid ester derivatives of ganciclovir,
including the di-valine, di-isoleucine, di-glycine and di-alanine
ester derivatives. International patent application WO95/09855,
published Apr. 13, 1995, discloses amino acid ester derivatives of
penciclovir, including the mono-valine and di-valine ester
derivatives.
[0012] DE 19526163, published Feb. 1, 1996 and U.S. Pat. No.
5,543,414 issued Aug. 6, 1996, disclose achiral amino acid esters
of ganciclovir.
[0013] European patent application EP 694 547, published Jan. 31,
1996, discloses the mono-L-valine ester of ganciclovir and its
preparation from di-valyl-ganciclovir.
[0014] European patent application EP 654 473, published May 24,
1995, discloses various bis amino acid ester derivatives of
9-[(1',2'-bishydroxymethyl)-cyclopropan-1'yl]methylguanine.
[0015] International patent application WO95/22330, published Aug.
24, 1995, discloses aliphatic esters, amino acid esters and mixed
acetate/valinate esters of the acyclic nucleoside
9-[3,3-dihydroxymethyl-- 4-hydroxy-but-1-yl]guanine. This reference
discloses that bioavailability is reduced when one of the valine
esters of the trivaline ester derivative is replaced with an
acetate ester.
BRIEF DESCRIPTION OF THE INVENTION
[0016] We have found that diester derivatives of H2G bearing
specific combinations of an amino acid ester and a fatty acid ester
are able to provide significantly improved oral bioavailability
relative to the parent compound (H2G). In accordance with a first
aspect of the invention there is thus provided novel compounds of
the formula I 2
[0017] wherein
[0018] a) R.sub.1 is --C(O)CH(CH(CH.sub.3).sub.2)NH.sub.2 or
--C(O)CH(CH(CH.sub.3)CH.sub.2CH.sub.3)NH.sub.2 and R.sub.2 is
--C(O)C.sub.3-C.sub.21 saturated or monounsaturated, optionally
substituted alkyl; or
[0019] b) R.sub.1 is --C(O)C.sub.3-C.sub.21 saturated or
monounsaturated, optionally substituted alkyl and R.sub.2 is
--C(O)CH(CH(CH.sub.3).sub.2)N- H.sub.2 or
--C(O)CH(CH(CH.sub.3)CH.sub.2CH.sub.3)NH.sub.2; and R.sub.3 is OH
or H;
[0020] or a pharmaceutically acceptable salt thereof.
[0021] The advantageous effect on oral bioavailability of the mixed
fatty acid and amino acid esters of the invention is particularly
unexpected in comparison to the oral bioavailability of the
corresponding fatty acid esters. Based on the results using a
urinary recovery assay (Table 1A) or a plasma drug assay (Table 1B)
of H2G from rats, neither the mono or di-fatty acid esters of H2G
provide any improvement in oral bioavailability relative to the
parent compound H2G. Indeed the di-stearate derivative provided
significantly lower bioavailability than the parent, indicating
that a stearate ester may be detrimental for improving oral
bioavailability of H2G. Converting one or both of the hydroxyls in
certain other acyclic nucleoside analogues to the corresponding
valine or di-valine ester has been reported to improve
bioavailability. Conversion of H2G to the coresponding mono- or
di-valyl ester derivatives produced similar improvement in
bioavailability relative to the parent compound. Given that fatty
acid derivatives of H2G are shown to be detrimental for improving
bioavailability, it was unexpected that a mixed amino acid/fatty
acid diester derivative of H2G would provide improved or comparable
oral bioavailability to that of the valine diester derivative of
H2G, based on urine recovery and plasma drug assays,
respectively.
1 TABLE 1A R.sub.1 group R.sub.2 group Bioavailability* hydrogen
hydrogen 8% hydrogen stearoyl 12% stearoyl stearoyl 1% valyl
hydrogen 29% valyl valyl 36% valyl stearoyl 56% *see Biological
Example 1 below for details
[0022]
2 TABLE 1B R.sub.1 group R.sub.2 group Bioavailability.sup.#
hydrogen hydrogen 3.8% hydrogen stearoyl 1.9% stearoyl stearoyl 0%
valyl hydrogen 31.3% valyl valyl 35.0% valyl stearoyl 29% .sup.#see
Biological Example 2 below for details
[0023] The invention also provides pharmaceutical compositions
comprising the compounds of Formula I and their pharmaceutically
acceptable salts in conjunction with a pharmaceutically acceptable
carrier or diluent. Further aspects of the invention include the
compounds of Formula I and their pharmaceutically acceptable salts
for use in therapy and the use of these compounds and salts in the
preparation of a medicament for the treatment or prophylaxis of
viral infection in humans or animals.
[0024] The compounds of the invention are potent antivirals,
especially against herpes infections, such as those caused by
Varicella zoster virus, Herpes simplex virus types 1 & 2,
Epstein-Barr virus, Herpes type 6 (HHV-6) and type 8 (HHV-8). The
compounds are particularly useful against Varicella zoster virus
infections such as shingles in the elderly including post herpetic
neuralgia or chicken pox in the young where the duration and
severity of the disease can be reduced by several days. Epstein
Barr virus infections amenable to treatment with the compounds
include infectious mononucleosis/glandular fever which has
previously not been treatable but which can cause many months of
scholastic incapacity amongst adolescents.
[0025] The compounds of the invention are also active against
certain retroviral infections, notably SIV, HIV-1 and HIV-2, and
against infections where a transactivating virus is indicated.
[0026] Accordingly a further aspect of the invention provides a
method for the prophylaxis or treatment of a viral infection in
humans or animals comprising the administration of an effective
amount of a compound of Formula I or its pharmaceutically
acceptable salt to the human or animal.
[0027] Advantageously group R.sub.3 is hydroxy or its tautomer
.dbd.O so that the base portion of the compounds of the invention
is the naturally occuring guanine, for instance in the event that
the side chain is cleaved in vivo. Alternatively, R.sub.3 may be
hydrogen thus defining the generally more soluble 6-deoxy
derivative which can be oxidised in vivo (e.g. by xanthine oxidase)
to the guanine form.
[0028] The compound of formula I may be present in racemic form,
that is a mixture of the 2R and 2S isomers. Preferably, however,
the compound of formula I has at least 70%, preferably at least 90%
R form, for example greater than 95%. Most preferably the compound
of formula I is enantiomerically pure R form.
[0029] Preferably the amino acid of group R.sub.1/R.sub.2 is
derived from an L-amino acid.
[0030] Preferably the fatty acid of group R.sub.1/R.sub.2 has in
total an even number of carbon atoms, in particular, decanoyl
(C.sub.10), lauryl (C.sub.12), myristoyl (C.sub.14), palmitoyl
(C.sub.16), stearoyl (C.sub.18) or eicosanoyl (C.sub.20). Other
useful R.sub.1/R.sub.2 groups include butyryl, hexanoyl, octanoyl
or behenoyl (C.sub.22). Further useful R.sub.1/R.sub.2 groups
include those derived from myristoleic, myristelaidic, palmitoleic,
palmitelaidic, n6-octadecenoic, oleic, elaidic, gandoic, erucic or
brassidic acids. Monounsaturated fatty acid esters typically have
the double bond in the trans configuration, preferably in the
.omega.-6, .omega.-9 or .omega.-11 position, dependent upon their
length. Preferably the R.sub.1/R.sub.2 group is derived from a
fatty acid which comprises a C.sub.9 to C.sub.17 saturated, or n:9
monounsaturated, alkyl.
[0031] The saturated or unsaturated fatty acid or R.sub.1/R.sub.2
may optionally be substituted with up to five similar or different
substituents independently selected from the group consisting of
such as hydroxy, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy,
C.sub.1-C.sub.6 alkoxy C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkanoyl, amino, halo, cyano, azido, oxo, mercapto and nitro, and
the like.
[0032] Most preferred compounds of the formula I are those where
R.sub.1 is --C(O)CH(CH(CH.sub.3).sub.2)NH.sub.2 or
--C(O)CH(CH(CH.sub.3)CH.sub.2C- H.sub.3)NH.sub.2 and R.sub.2 is
--C(O)C.sub.9-C.sub.17 saturated alkyl.
[0033] The term "lower alkyl" as used herein refers to straight or
branched chain alkyl radicals containing from 1 to 7 carbon atoms
including, but not limited to, methyl, ethyl, n-propyl, iso-propyl,
n-butyl, iso-butyl, sec-butyl, t-butyl, n-pentyl, 1-methylbutyl,
2,2-dimethylbutyl, 2-methylpentyl, 2,2-dimethylpropyl, n-hexyl and
the like.
[0034] The term "alkanoyl" as used herein refers to R.sub.20C(O)--
wherein R.sub.20 is a loweralkyl group.
[0035] The term "alkoxy" as used herein refers to R.sub.21O--
wherein R.sub.21 is a loweralkyl group.
[0036] The term "alkoxyalkyl" as used herein refers to an alkoxy
group appended to a loweralkyl radical.
[0037] The term "N-protecting group" or "N-protected" as used
herein refers to those groups intended to protect the N-terminus of
an amino acid or peptide or to protect an amino group against
undesirable reactions during synthetic procedures. Commonly used
N-protecting groups are disclosed in Greene, "Protective Groups in
Organic Synthesis" (John Wiley & Sons, New York, 1981), which
is hereby incorporated by reference. N-protecting groups include
acyl groups such as formyl, acetyl, propionyl, pivaloyl,
t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoracetyl,
trichloroacetyl, phthalyl, o-nitrophenoxyacetyl,
.alpha.-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl,
4-nitrobenzoyl, and the like; sulfonyl groups such as
benzenesulfonyl, p-toluenesulfonyl, and the like, carbamate forming
groups such as benzyloxycarbonyl, p-chlorobenzyloxy-carbonyl,
p-methoxybenzyloxycarbonyl- , p-nitrobenzyloxycarbonyl,
2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,
3,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,
2-nitro-4,5-dimethoxybenzyloxycarbonyl,
3,4,5-trimethoxybenzyloxycarbonyl,
1-(p-biphenylyl)-1-methylethoxycarbony- l,
.alpha.,.alpha.-dimethyl-3,5-dimethoxybenzyloxycarbonyl,
benzhydryloxycarbonyl, t-butoxycarbonyl,
diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl,
methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl,
phenoxycarbonyl, 4-nitrophenoxycarbonyl,
fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,
adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl,
and the like; alkyl gropus such as benzyl, triphenylmethyl,
benzyloxymethyl and the like; and silyl groups such as
trimethylsilyl and the like. Favoured N-protecting groups include
formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl,
benzyl, t-butoxycarbonyl (BOC) and benzyloxycarbonyl (Cbz).
[0038] The term "O-protecting group" or "hydroxy-protecting group"
or "--OH protecting group" as used herein refers to a substituent
which protects hydroxyl groups against undesirable reactions during
synthetic procedures such as those O-protecting groups disclosed in
Greene, "Protective Groups In Organic Synthesis," (John Wiley &
Sons, New York (1981)). O-protecting groups comprise substituted
methyl ethers, for example, methoxymethyl, benzyloxymethyl,
2-methoxyethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, t-butyl,
benzyl and triphenylmethyl; tetrahydropyranyl ethers; substituted
ethyl ethers, for example, 2,2,2-trichloroethyl; silyl ethers, for
example, trimethylsilyl, t-butyldimethylsilyl and
t-butyldiphenylsilyl; and esters prepared by reacting the hydroxyl
group with a carboxylic acid, for example, acetate, propionate,
benzoate and the like.
[0039] The term "activated ester derivative" as used herein refers
to acid halides such as acid chlorides, and activated esters
including, but not limited to, formic and acetic acid derived
anhydrides, anhydrides derived from alkoxycarbonyl halides such as
isobutyloxycarbonylchloride and the like, N-hydroxysuccinimide
derived esters, N-hydroxyphthalimide derived esters,
N-hydroxybenzotriazole derived esters, N-hydroxy-5-norbornene-2,3-
-dicarboxamide derived esters, 2,4,5-trichlorophenyl derived
esters, sulfonic acid derived anhydrides (for example,
p-toluenesulonic acid derived anhydrides and the like) and the
like.
[0040] Preferred compounds of formula I include:
[0041]
(R)-9-[2-(butyryloxymethyl)-4-(L-isoleucyloxy)butyl]guanine,
[0042]
(R)-9-[2-(4-acetylbutyryloxymethyl)-4-(L-isoleucyloxy)butyl]guanine-
,
[0043]
(R)-9-[2-(hexanoyloxymethyl)-4-(L-isoleucyloxy)butyl]guanine,
[0044]
(R)-9-[4-(L-isoleucyloxy)-2-(octanoyloxymethyl)butyl]guanine,
[0045]
(R)-9-[4-(L-isoleucyloxy)-2-(decanoyloxymethyl)butyl]guanine,
[0046]
(R)-9-[4-(L-isoleucyloxy)-2-(dodecanoyloxymethyl)butyl]guanine,
[0047]
(R)-9-[4-(L-isoleucyloxy)-2-(tetradecanoyloxymethyl)butyl]guanine,
[0048]
(R)-9-[4-(L-isoleucyloxy)-2-(hexadecanoyloxymethyl)butyl]guanine,
[0049]
(R)-9-[4-(L-isoleucyloxy)-2-(octadecanoyloxymethyl)butyl]guanine,
[0050]
(R)-9-[2-(eicosanoyloxymethyl)-4-(L-isoleucyloxy)butyl]guanine,
[0051]
(R)-9-[2-(docosanoyloxymethyl)-4-(L-isoleucyloxy)butyl]guanine,
[0052]
(R)-9-[4-(L-isoleucyloxy)-2-((9-tetradecenoyl)oxymethyl)butyl]guani-
ne,
[0053]
(R)-9-[2-((9-hexadecenoyl)oxymethyl)-4-(L-isoleucyloxy)butyl]guanin-
e,
[0054]
(R)-9-[4-(L-isoleucyloxy)-2-((6-octadecenoyl)oxymethyl)butyl]guanin-
e,
[0055]
(R)-9-[4-(L-isoleucyloxy)-2-((9-octadecenoyl)oxymethyl)-butyl]guani-
ne,
[0056]
(R)-9-[2-((11-eicosanoyl)-oxymethyl)-4-(L-isoleucyloxy)butyl]guanin-
e,
[0057]
(R)-9-[2-((13-docosenoyl)-oxymethyl)-4-(L-isoleucyloxy)butyl]guanin-
e,
[0058]
(R)-2-amino-9-[2-(butyryloxymethyl)-4-(L-isoleucyloxy)butyl]purine,
[0059]
R)-2-amino-9-[2-(4-acetylbutyryloxymethyl)-4-(L-isoleucyloxy)butyl]-
purine,
[0060]
(R)-2-amino-9-[2-(hexanoyloxymethyl)-4-(L-isoleucyloxy)butyl]purine-
,
[0061]
(R)-2-amino-9-[4-(L-isoleucyloxy)-2-(octanoyloxymethyl)butyl]purine-
,
[0062]
(R)-2-amino-9-[4-(L-isoleucyloxy)-2-(decanoyloxymethyl)butyl]purine-
,
[0063]
(R)-2-amino-9-[4-(L-isoleucyloxy)-2-(dodecanoyloxymethyl)butyl]puri-
ne,
[0064]
(R)-2-amino-9-[4-(L-isoleucyloxy)-2-(tetradecanoyloxymethyl)butyl]p-
urine,
[0065]
(R)-2-amino-9-[4-(L-isoleucyloxy)-2-(hexadecanoyloxymethyl)butyl]pu-
rine,
[0066]
(R)-2-amino-9-[4-(L-isoleucyloxy)-2-(octadecanoyloxymethyl)butyl]pu-
rine,
[0067]
(R)-2-amino-9-[4-(L-isoleucyloxy)-2-(eicosanoyloxymethyl)butyl]puri-
ne,
[0068]
(R)-2-amino-9-[2-(eicosanoyloxymethyl)-4-(L-isoleucyloxy)butyl]puri-
ne,
[0069]
(R)-2-amino-9-[2-(docosanoyloxymethyl)-4-(L-isoleucyloxy)butyl]puri-
ne
[0070]
(R)-2-amino-9-[4-(L-isoleucyloxy)-2-((9-tetradecenoyl)oxymethyl)but-
yl]purine,
[0071]
(R)-2-amino-9-[2-((9-hexadecenoyl)oxymethyl)-4-(L-isoleucyloxy)buty-
l]purine,
[0072]
(R)-2-amino-9-[4-(L-isoleucyloxy)-2-((6-octadecenoyl)oxymethyl)buty-
l]purine,
[0073]
(R)-2-amino-9-[4-(L-isoleucyloxy)-2-((9-octadecenoyl)oxymethyl)buty-
l]purine,
[0074]
(R)-2-amino-9-[2-((11-eicosanoyl)oxymethyl)-4-(L-isoleucyloxy)butyl-
]purine, or
[0075]
(R)-2-amino-9-[2-((13-docosenoyl)oxymethyl)-4-(L-isoleucyloxy)butyl-
]purine,
[0076] or a pharmaceutically accepable salt thereof.
[0077] Further preferred compounds include:
[0078] (R)-9-[2-(butyryloxymethyl)-4-(L-valyloxy)butyl]guanine,
[0079]
(R)-9-[2-(4-acetylbutyryloxymethyl)-4-(L-valyloxy)butyl]guanine,
[0080]
(R)-9-[2-(hexanoyloxymethyl)-4-(L-valyloxy)butyl]guanine,
[0081]
(R)-9-[2-(octanoyloxymethyl)-4-(L-valyloxy)butyl]guanine,
[0082]
(R)-9-[2-(decanoyloxymethyl)-4-(L-valyloxy)butyl]guanine,
[0083]
(R)-9-[2-(dodecanoyloxymethyl)-4-(L-valyloxy)butyl]guanine,
[0084]
(R)-9-[2-(tetradecanoyloxymethyl-4-(L-valyloxy)butyl]guanine,
[0085]
(R)-9-[2-hexadecanoyloxymethyl)-4-(L-valyloxy)butyl]guanine,
[0086]
(R)-9-[2-(octadecanoyloxymethyl)-4-(L-valyloxy)butyl]guanine,
[0087]
(R)-9-[2-(eicosanoyloxymethyl)-4-(L-valyloxy)butyl]guanine,
[0088]
(R)-9-[2-(eicosanoyloxymethyl)-4-(L-valyloxy)butyl]guanine,
[0089]
(R)-9-[2-(docosanoyloxymethyl)-4-(L-valyloxy)butyl]guanine,
[0090]
(R)-9-[2-((9-tetradecenoyl)oxymethyl)-4-(L-valyloxy)butyl]guanine,
[0091]
(R)-9-[2-((9-hexadecenoyl)oxymethyl)-4-(L-valyloxy)butyl]guanine,
[0092]
(R)-9-[2-((6-octadecenoyl)oxymethyl)-4-(L-valyloxy)butyl]guanine,
[0093]
(R)-9-[2-((9-octadecenoyl)oxymethyl)-4-(L-valyloxy)-butyl]guanine,
[0094]
(R)-9-[2-((1-eicosanoyl)oxymethyl)-4-(L-valyloxy)butyl]guanine,
[0095]
(R)-9-[2-((13-docosenoyl)oxymethyl)-4-(L-valyloxy)butyl]guanine,
[0096]
(R)-2-amino-9-[2-(butyryloxymethyl)-4-(L-valyloxy)butyl]purine,
[0097]
(R)-2-amino-9-[2-(4-acetylbutyryloxymethyl)-4-(L-valyloxy)butyl]pur-
ine,
[0098]
(R)-2-amino-9-[2-(hexanoyloxymethyl)-4-(L-valyloxy)butyl]purine,
[0099]
(R)-2-amino-9-[2-(octanoyloxymethyl)-4-(L-valyloxy)butyl]purine,
[0100]
(R)-2-amino-9-[2-(decanoyloxymethyl)-4-(L-valyloxy)butyl]purine,
[0101]
(R)-2-amino-9-[2-(dodecanoyloxymethyl)-4-(L-valyloxy)butyl]purine,
[0102]
(R)-2-amino-9-[2-(tetradecanoyloxymethyl)-4-(L-valyloxy)butyl]purin-
e,
[0103]
(R)-2-amino-9-[2-(hexadecanoyloxymethyl)-4-(L-valyloxy)butyl]purine-
,
[0104]
(R)-2-amino-9-[2-(octadecanoyloxymethyl)-4-(L-valyloxy)-butyl]purin-
e,
[0105]
(R)-2-amino-9-[2-(eicosanoyloxymethyl)-4-(L-valyloxy)butyl]purine,
[0106]
(R)-2-amino-9-[2-(docosanoyloxymethyl)-4-(L-valyloxy)butyl]purine,
[0107]
(R)-2-amino-9-[2-((9-tetradecenoyl)oxymethyl)-4-(L-valyloxy)butyl]p-
urine,
[0108]
(R)-2-amino-9-[2-((9-hexadecenoyl)oxymethyl)-4-(L-valyloxy)butyl]pu-
rine,
[0109]
(R)-2-amino-9-[2-((6-octadecenoyl)oxymethyl)-4-(L-valyloxy)butyl]pu-
rine,
[0110]
(R)-2-amino-9-[2-((9-octadecenoyl)oxymethyl)-4-(L-valyloxy)-butyl]p-
urine,
[0111]
(R)-2-amino-9-[2-((11-eicosenoyl)-oxymethyl)-4-(L-valyloxy)butyl]pu-
rine, or
[0112]
(R)-2-amino-9-[2-((13-docosenoyl)-oxymethyl)-4-(L-valyloxy)butyl]pu-
rine;
[0113] or a pharmaceutically acceptable salt thereof.
[0114] Other preferred compounds of formula I include:
[0115] (R)-9-[4-(butyryloxy)-2-(L-valyloxymethyl)butyl]guanine,
[0116]
(R)-9-[4-(4-acetylbutyryloxy)-2-(L-valyloxymethyl)butyl]guanine,
[0117]
(R)-9-[4-(hexanoyloxy)-2-(L-valyloxymethyl)butyl]guanine,
[0118]
(R)-9-[4-(octanoyloxy)-2-(L-valyloxymethyl)butyl]guanine,
[0119]
(R)-9-[4-(decanoyloxy)-2-(L-valyloxymethyl)butyl]guanine,
[0120]
(R)-9-[4-(dodecanoyloxy)-2-(L-valyloxymethyl)butyl]guanine,
[0121]
(R)-9-[4-(tetradecanoyloxy)-2-(L-valyloxymethyl)butyl]guanine,
[0122]
(R)-9-[4-hexadecanoyloxy)-2-(L-valyloxymethyl)butyl]guanine,
[0123]
(R)-9-[4-(octadecanoyloxy)-2-(L-valyloxymethyl)butyl]guanine,
[0124]
(R)-9-[4-(eicosanoyloxy)-2-(L-valyloxymethyl)butyl]guanine,
[0125]
(R)-9-[4-(docosanoyloxy)-2-(L-valyloxymethyl)butyl]guanine,
[0126]
(R)-9-[4-((9-tetradecenoyl)oxy)-2-(L-valyloxymethyl)butyl]guanine,
[0127]
(R)-9-[4-((9-hexadecenoyl)oxy)-2-(L-valyloxymethyl)butyl]guanine,
[0128]
(R)-9-[4-((6-octadecenoyl)oxy)-2-(L-valyloxymethyl)butyl]guanine,
[0129]
(R)-9-[4-((9-octadecenoyl)oxy)-2-(L-valyloxymethyl)-butyl]guanine,
[0130]
(R)-9-[4-((11-eicosenoyl)oxy)-2-(L-valyloxymethyl)butyl]guanine,
[0131]
(R)-9-[4-((13-docosenoyl)-oxy)-2-(L-valyloxymethyl)butyl]guanine,
[0132]
(R)-2-amino-9-[4-(butyryloxy)-2-(L-valyloxymethyl)butyl]purine,
[0133]
(R)-2-amino-9-[4-(4-acetylbutyryloxy)-2-(L-valyloxymethyl)butyl]pur-
ine,
[0134]
(R)-2-amino-9-[4-(hexanoyloxy)-2-(L-valyloxymethyl)butyl]purine,
[0135]
(R)-2-amino-9-[4-(octanoyloxy)-2-(L-valyloxymethyl)butyl]purine,
[0136]
(R)-2-amino-9-[4-(decanoyloxy)-2-(L-valyloxymethyl)butyl]purine,
[0137]
(R)-2-amino-9-[4-(dodecanoyloxy)-2-(L-valyloxymethyl)butyl]purine,
[0138]
(R)-2-amino-9-[4-(tetradecanoyloxy)-2-(L-valyloxymethyl)butyl]purin-
e,
[0139]
(R)-2-amino-9-[4-(hexadecanoyloxy)-2-(L-valyloxymethyl)butyl]purine-
,
[0140]
(R)-2-amino-9-[4-(octadecanoyloxy)-2-(L-valyloxymethyl)-butyl]purin-
e,
[0141]
(R)-2-amino-9-[4-(eicosanoyloxy)-2-(L-valyloxymethyl)butyl]purine,
[0142]
(R)-2-amino-9-[4-(docosanoyloxy)-2-(L-valyloxymethyl)butyl]purine,
[0143]
(R)-2-amino-9-[4-((9-tetradecenoyl)oxy)-2-(L-valyloxymethyl)butyl]p-
urine,
[0144]
(R)-2-amino-9-[4-((9-hexadecenoyl)oxy)-2-(L-valyloxymethyl)butyl]pu-
rine,
[0145]
(R)-2-amino-9-[4-((6-octadecenoyl)oxy)-2-(L-valyloxymethyl)butyl]pu-
rine,
[0146]
(R)-2-amino-9-[4-((9-octadecenoyl)oxy)-2-(L-valyloxymethyl)butyl]pu-
rine,
[0147]
(R)-2-amino-9-[4-((11-eicosenoyl)oxy)-2-(L-valyloxymethyl)butyl]pur-
ine, or
[0148]
(R)-2-amino-9-[4-((13-docosenoyl)oxy)-2-(L-valyloxymethyl)butyl]pur-
ine,
[0149] or a pharmaceutically acceptable salt thereof.
[0150] The compounds of formula I can form salts which form an
additional aspect of the invention. Appropriate pharmaceutically
acceptable salts of the compounds of formula I include salts of
organic acids, especially carboxylic acids, including but not
limited to acetate, trifluoroacetate, lactate, gluconate, citrate,
tartrate, maleate, malate, pantothenate, isethionate, adipate,
alginate, aspartate, benzoate, butyrate, digluconate,
cyclopentanate, glucoheptanate, glycerophosphate, oxalate,
heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate,
3-phenylpropionate, picrate, pivalate, proprionate, tartrate,
lactobionate, pivolate, camphorate, undecanoate and succinate,
organic sulphonic acids such as methanesulphonate,
ethanesulphonate, 2-hydroxyethane sulphonate, camphorsulphonate,
2-napthalenesulphonate, benzenesulphonate,
p-chlorobenzenesulphonate and p-toluenesulphonate; and inorganic
acids such as hydrochloride, hydrobromide, hydroiodide, sulphate,
bisulphate, hemisulphate, thiocyanate, persulphate, phosphoric and
sulphonic acids. Hydrochloric acid salts are convenient.
[0151] The compounds of Formula I may be isolated as the hydrate.
The compounds of the invention may be isolated in crystal form,
preferably homogenous crystals, and thus an additional aspect of
the invention provides the compounds of Formula I in substantially
pure crystalline form, comprising >70%, preferably >90%
homogeneous crystalline material, for example >95% homogeneous
crystalline material.
[0152] The compounds of the invention are particularly suited to
oral administration, but may also be administered rectally,
vaginally, nasally, topically, transdermally or parenterally, for
instance intramuscularly, intravenously or epidurally. The
compounds may be administered alone, for instance in a capsule, but
will generally be administered in conjunction with a
pharmaceutically acceptable carrier or diluent. The invention
extends to methods for preparing a pharmaceutical composition
comprising bringing a compound of Formula I or its pharmaceutically
acceptable salt in conjunction or association with a
pharmaceutically acceptable carrier or vehicle.
[0153] Oral formulations are conveniently prepared in unit dosage
form, such as capsules or tablets, employing conventional carriers
or binders such as magnesium stearate, chalk, starch, lactose, wax,
gum or gelatin. Liposomes or synthetic or natural polymers such as
HPMC or PVP may be used to afford a sustained release formulation.
Alternatively the formulation may be presented as a nasal or eye
drop, syrup, gel or cream comprising a solution, suspension,
emulsion, oil-in-water or water-in-oil preparation in conventional
vehicles such as water, saline, ethanol, vegetable oil or
glycerine, optionally with flavourant and/or preservative and/or
emulsifier.
[0154] The compounds of the invention may be administered at a
daily dose generally in the range 0.1 to 200 mg/kg/day,
advantageously, 0.5 to 100 mg/kg/day, more preferably 10 to 50
mg/kg/day, such as 10 to 25 mg/kg/day. A typical dosage rate for a
normal adult will be around 50 to 500 mg, for example 300 mg, once
or twice per day for herpes infections and 2 to 10 times this
dosage for HIV infections.
[0155] As is prudent in antiviral therapy, the compounds of the
invention can be administered in combination with other antiviral
agents, such as acyclovir, valcyclovir, penciclovir, famciclovir,
ganciclovir and its prodrugs, cidofovir, foscarnet and the like for
herpes indications and AZT, ddI, ddC, d4T, 3TC, foscarnet,
ritonavir, indinavir, saquinavir, delaviridine, Vertex VX 478,
Agouron AG1343 and the like for retroviral indications.
[0156] The compounds of the invention can be prepared de novo or by
esterification of the H2G parent compound which is prepared, for
example, by the synthesis methodology disclosed in European Patent
EP 343 133, which is incorporated herein by reference.
[0157] A typical reaction scheme for the preparation of H2G is
depicted below: 3
[0158] The condensation in step 1 is typically carried out with a
base catalyst such as NaOH or Na.sub.2CO.sub.3 in a solvent such as
DMF. Step 2 involves a reduction which can be performed with
LiBH.sub.4/tetrahydrofuran in a solvent such as t-BuOH. The
substitution in step 3 of the chlorine with an amino group can be
performed under pressure with ammonia. Step 4 employs adenosine
deaminase which can be conveniently immobilized on a solid support.
Cooling the reaction mixture allows unreacted isomeric precursor to
remain in solution thereby enhancing purity.
[0159] Starting materials for compounds of the invention in which
R.sub.3 is hydrogen may be prepared as shown in European Patent EP
186 640, the contents of which are incorporated herein by
reference. These starting materials may be acylated as described
for H2G below, optionally after protecting the purine 2-amino group
with a conventional N-protecting group as defined above, especially
BOC (t-BuO--CO--), Z (BnO--CO--) or Ph.sub.3C--.
[0160] The compounds of the invention may be prepared from H2G as
described below in Schemes A and B.
[0161] A. Direct Acylation Method 4
[0162] Scheme A depicts the preparation of compounds in which
R.sub.2 is derived from the amino acid and R.sub.2 is derived from
the fatty acid, but the converse scheme is applicable to compounds
where R.sub.1 is derived from the fatty acid and R.sub.2 is derived
from the amino acid ester. In the variant specifically depicted in
scheme A above, G is guanine or 6-deoxyguanine, PG is an optional
N-protecting group or hydrogen, R.sub.1* is the valine or
isoleucine side chain and R.sub.2* is the fatty acid chain. H2G is
depicted above as a starting material but this of course may be
optionally protected at R.sub.3 or the 2 position of the purine
with conventional N-protecting groups (not shown). The H2G
(derivative) reacts in the first step with an activated R.sub.1
.alpha.-amino acid derivative, as further described below, in a
solvent such as dimethylformamide or pyridine, to give a
monoacylated product. The R.sub.1 .alpha.-amino acid may be
suitably N-protected with N-BOC or N-CBz or the like. Under
controlled conditions, the first acylation can be made to
predominantly take place at the side chain 4-hydroxy group on the
side chain of H2G. These controlled conditions can be achieved, for
example, by manipulating the reagent concentrations or rate of
addition, especially of the acylating agent, by lowering the
temperature or by the choice of solvent. The reaction can be
followed by TLC to monitor the controlled conditions.
[0163] After purification, the R.sub.1 monoacylated compounds are
further acylated on the side chain 2-CH.sub.2OH group with the
appropriate activated fatty acid derivative to give diacylated
products using similar procedures as for the first esterification
step. The diester products are subsequently subjected to a
conventional deprotection treatment using for example
trifluoroacetic acid, HCl(aq)/dioxane or hydrogenation in the
presence of catalyst to give the desired compound of Formula I. The
compound may be in salt form depending on the deprotection
conditions.
[0164] The activated R.sub.1/R.sub.2 acid derivative used in the
various acylations may comprise e.g. the acid halide, acid
anhydride, activated acid ester or the acid in the presence of
coupling reagent, for example dicyclohexylcarbodiimide, where
"acid" in each case represents the corresponding R.sub.1/R.sub.2
amino acid or the R.sub.1/R.sub.2 fatty acid. Representative
activated acid derivatives include the acid chloride, formic and
acetic acid derived mixed anhydrides, anhydrides derived from
alkoxycarbonyl halides such as isobutyloxycarbonylchloride and the
like, N-hydroxysuccinamide derived esters, N-hydroxyphthalimide
derived esters, N-hydroxy-5-norbornene-2,3-dicarboxamide derived
esters, 2,4,5-trichlorophenol derived esters, sulfonic acid derived
anhydrides (for example, p-toluenesulonic acid derived anhydrides
and the like) and the like.
[0165] B. Via Protection of the Side Chain 4-Hydroxy Group: 5
[0166] wherein G, PG, R.sub.1* and R.sub.2* are as described for
scheme A.
[0167] Scheme B has been exemplified with reference to the
preparation of a compound where R.sub.1 is derived from an amino
acid and R.sub.2 is derived from the fatty acid ester, but a
converse scheme will be applicable to compounds where R.sub.2 is
derived from the amino acid and R.sub.1 is derived from the fatty
acid. This scheme relies on regioselective protection of the H2G
side chain 4-hydroxy group with a bulky protecting group. In scheme
B above this is depicted as t-butyldiphenylsilyl, but other
regioselective protecting groups such as trityl,
9-(9-phenyl)xanthenyl, 1,1-bis(4-methylphenyl)-1'-pyrenylmethyl may
also be appropriate. The resulting product is acylated at the side
chain 2-hydroxymethyl group using analogous reagents and procedures
as described in scheme A above, but wherein the activated acid
derivative is the R.sub.2 fatty acid, for example, myristic,
stearic, oleic, elaidic acid chloride and the like. The thus
monoacylated compounds are subjected to appropriate deprotection
treatment to remove the side chain 4-hydroxy protecting group which
can be done in a highly selective manner with such reagents,
depending on the regioselective protecting group, as HF/pyridine
and the like and manipulation of the reaction conditions, viz
reagent concentration, speed of addition, temperature and solvent
etc, as elaborated above. The then free side chain 4-hydroxy group
is acylated with the activated .alpha.-amino acid in a similar way
as described in scheme A above.
[0168] Additional techniques for introducing the amino acid ester
of R.sub.1/R.sub.2, for instance in schemes A, B, C, D or E herein
include the 2-oxa-4-aza-cycloalkane-1,3-dione method described in
International patent application No. WO 94/29311.
[0169] Additional techniques for introducing the fatty acid ester
of R.sub.1/R.sub.2, for instance in schemes A, B, C, D or E herein
include the enzymatic route described in Preparative
Biotransformations 1.11.8 (Ed S M Roberts, J Wiley and Son, NY,
1995) with a lipase such as SP 435 immobilized Candida antarcticus
(Novo Nordisk), porcine pancreatic lipase or Candida rugosa lipase.
Enzymatic acylation is especially convenient where it is desired to
avoid N-protection and deprotection steps on the other acyl group
or the purine 2-amine.
[0170] An alternative route to compounds of Formula I in which
R.sub.3 is hydrogen is to 6-activate the correponding guanine
compound of Formula I (wherein the amino acid ester moiety of
R.sub.1/R.sub.2 is optionally protected with conventional
N-protecting groups such as BOC) with an activating group such as
halo. The thus activated 6-purine is subsequently reduced to
purine, for instance with a palladium catalyst and deprotected to
the desired 6-deoxy H2G di-ester.
[0171] A further aspect of the invention thus provides a method for
the preparation of the compounds of formula I comprising
[0172] a) optionally N-protecting the purine 2 and/or 6 positions
of a compound of formula I wherein R.sub.1 and R.sub.2 are each
hydrogen;
[0173] b) regioselectively acylating the compound of Formula I at
the side chain 4-hydroxy group with either
[0174] i) an optionally N-protected valine or isoleucine group,
[0175] ii) an optionally substituted, saturated or monounsaturated
C.sub.3-C.sub.21COOH derivative, or
[0176] iii) a regioselective protecting group;
[0177] c) acylating at the side chain 2-hydroxymethyl group
with
[0178] i) an optionally N-protected valine or isoleucine
derivative, or
[0179] ii) an optionally substituted, saturated or monounsaturated
C.sub.3-C.sub.21COOH derivative;
[0180] d) replacing the regioselective protecting group at R.sub.1,
if present, with
[0181] i) an optionally N-protected valine or isoleucine
derivative; or
[0182] ii) an optionally substituted, saturated or monounsaturated
C.sub.3-C.sub.21COOH derivative; and
[0183] e) deprotecting the resulting compound as necessary.
[0184] Schemes A and B above employ selective acylation to stepwise
add the amino acid and fatty acid esters. An alternative process
for the preparation of the compounds of formula I starts with a
diacylated H2G derivative, wherein both the acyl groups are the
same, and employs selective removal of one of the acyl groups to
obtain a monoacyl intermediate which is then acylated with the
second, differing, acyl group in the same manner as Schemes A and B
above.
[0185] Accordingly a further aspect of the invention provides a
method for the preparation of a compound of the formula I, as
defined above, which method comprises
[0186] A) the monodeacylation of a diacylated compound
corresponding to formula I wherein R.sub.1 and R.sub.2 are both a
valyl or isoleucyl ester (which is optionally N-protected) or
wherein R.sub.1 and R.sub.2 are both --C(.dbd.O)C.sub.3-C.sub.21
saturated or monounsaturated, optionally substituted alkyl; and
[0187] B) acylating the thus liberated side chain 4-hydroxy or side
chain 2-hydroxymethyl group with the corresponding valyl, isoleucyl
or --C(.dbd.O)C.sub.3-C.sub.21 saturated or monounsaturated,
optionally substituted alkyl; and
[0188] C) deprotecting as necessary.
[0189] This alternative process has the advantage that the
preparation of the diacylated H2G derivative is facile and requires
little or no purification steps. Selective removal of one only of
the acyl groups of a diacylated H2G derivative can be achieved by
manipulating the reaction conditions, in particular the
temperature, rate of reactant addition and choice of base.
[0190] Compounds amenable to this alternative synthesis route are
thus of the formula: 6
[0191] wherein R.sub.1 and R.sub.2 are valyl or isoleucyl (which
are optionally N-protected) or a --C(.dbd.O)C.sub.3-C.sub.21
saturated or monounsaturated, optionally substituted alkyl; and
R.sub.3 is OH or H.
[0192] For ease of synthesis in this alternative route, it is
preferred that R.sub.1 and R.sub.2 are both initially identical and
are most preferably the same amino acid ester. Such a di-amino acid
ester will generally be N-protected during its preparation and may
be used directly in this condition in the selective deacylation
step. Alternatively, such an N-protected di-aminoacylated H2G
derivative may be deprotected and optionally reprotected, as
described below. The unprotected di-aminoacyl H2G derivative thus
comprises one of the following compounds:
[0193]
(R)-9-[2-(L-isoleucyloxymethyl)-4-(L-isoleucyloxy)butyl]guanine,
[0194] (R)-9-[2-(L-valyloxymethyl)-4-(L-valyloxy)butyl]guanine,
[0195]
(R)-2-amino-9-[4-(L-isoleucyloxy)-2-(L-isoleucyloxymethyl)butyl]pur-
ine, and
[0196]
(R)-2-amino-9-[4-(L-valyloxy)-2-(L-valyloxymethyl)butyl]purine.
[0197] These unprotected H2G diacylated derivatives can be directly
subject to selective deacylation of one of the acyl groups
(typically the side chain 4-position acyl) followed by enzymatic
acylation of the liberated 4-hydroxy as described above.
Alternatively, the unprotected H2G diacylated derivative can be
re-protected and then subjected to the selective deacylation,
followed in turn by conventional acylation with the fatty acid
ester, as described in Schemes A and B. Conveniently, such a
reprotection step is done with a different N-protecting group,
having properties appropriate to the subsequent acylation. For
example, it is convenient to employ a lipophilic N-protecting
group, such as Fmoc when preparing a di-amino acid H2G derivative,
as the lipophilic nature of the protecting group assists with
separation of the acylated products. On the other hand, the
lipophilic nature of Fmoc is of less utility when conducting an
acylation with a fatty acid, and thus it is convenient to reprotect
a diacylated H2G with an alternative N-protecting group such as
BOC.
[0198] It will also be apparent that the preparation of the
compounds of formula I can commence with the novel monoacylated
intermediates of step b i), ii) or iii) in the above defined first
method aspect of the invention. These compounds are thus of the
formula: 7
[0199] wherein one of R.sub.1 and R.sub.2 is
[0200] i) --C(O)CH(CH(CH.sub.3).sub.2)NH.sub.2 or
--C(O)CH(CH(CH.sub.3)CH.- sub.2CH.sub.3)NH.sub.2,
[0201] ii) a --C(.dbd.O)C.sub.3-C.sub.21 saturated or
monounsaturated, optionally substituted alkyl, or
[0202] iii) a regioselective protecting group;
[0203] the other of R.sub.1 and R.sub.2 is hydrogen; and
[0204] R.sub.3 is OH or H.
[0205] Useful compounds thus include:
[0206]
(R)-9-[2-hydroxymethyl-4-(t-butyldiphenylsilyl)butyl]guanine,
[0207] (R)-9-[2-hydroxymethyl-4-(trityloxy)butyl]guanine,
[0208]
(R)-9-[2-hydroxymethyl-4-(9-(9-phenyl)xanthenyloxy)butyl]guanine,
[0209]
(R)-9-[2-hydroxymethyl-4-(1,1-bis(4-methylphenyl)-1'-pyrenylmethylo-
xy)butyl]guanine,
[0210] (R)-9-[2-hydroxymethyl-4-(decanoyloxy)butyl]guanine,
[0211] (R)-9-[2-hydroxymethyl)-4-(dodecanoyloxy)butyl]guanine,
[0212]
(R)-9-[2-hydroxymethyl-4-(tetradecanoyloxy)butyl]guanine,
[0213]
(R)-9-[2-hydroxymethyl)-4-(hexadecanoyloxy)butyl]guanine,
[0214] (R)-9-[2-hydroxymethyl-4-(octadecanoyloxy)butyl]guanine,
[0215] (R)-9-[2-hydroxymethyl)-4-(eicosanoyloxy)butyl]guanine,
[0216] (R)-9-[2-hydroxymethyl-4-(docosanoyloxy)butyl]guanine,
[0217] (R)-9-[4-hydroxy-2-(decanoyloxymethyl)butyl]guanine,
[0218] (R)-9-[4-hydroxy-2-(dodecanoyloxymethyl) butyl]guanine,
[0219]
(R)-9-[4-hydroxy-2-(tetradecanoyloxymethyl)butyl]guanine,
[0220] (R)-9-[4-hydroxy-2-(hexadecanoyloxymethyl)butyl]guanine,
[0221] (R)-9-[4-hydroxy-2-(octadecanoyloxymethyl)butyl]guanine,
[0222] (R)-9-[4-hydroxy-2-(eicosanoyloxymethyl)butyl]guanine,
[0223] (R)-9-[4-hydroxy-2-(docosanoyloxymethyl)butyl]guanine,
[0224] (R)-9-[2-hydroxymethyl-4-(L-valyloxy)butyl]guanine,
[0225] (R)-9-[2-hydroxymethyl)-4-(L-isoleucyloxy)butyl]guanine,
[0226] (R)-9-[4-hydroxy-2-(L-isoleucyloxymethyl)butyl]guanine,
[0227] (R)-9-[4-hydroxy-2-(L-valyloxymethyl)butyl]guanine.
[0228]
(R)-2-amino-9-[2-hydroxymethyl-4-(L-valyloxy)butyl]purine,
[0229]
(R)-2-amino-9-[2-hydroxymethyl)-4-(L-isoleucyloxy)butyl]purine,
[0230]
(R)-2-amino-9-[4-hydroxy-2-(L-isoleucyloxymethyl)butyl]purine,
and
[0231]
(R)-2-amino-9-[4-hydroxy-2-(L-valyloxymethyl)butyl]purine.
[0232] Regioselectively protected, sidechain 4-hydroxy
intermediates from step c) of the above described first method
aspect of the invention are also novel compounds. Useful compounds
thus include:
[0233]
(R)-9-[2-decanoyloxymethyl-4-(t-butyldiphenylsilyl)butyl]guanine,
[0234]
(R)-9-[2-dodecanoyloxymethyl-4-(t-butyldiphenylsilyl)butyl]guanine,
[0235]
(R)-9-[2-tetradecanoyloxymethyl-4-(t-butyldiphenylsilyl)butyl]guani-
ne,
[0236]
(R)-9-[2-hexadecanoyloxymethyl-4-(t-butyldiphenylchlorosilane)butyl-
]guanine,
[0237]
(R)-9-[2-octadecanoyloxymethyl-4-(t-butyldiphenylsilyl)butyl]guanin-
e,
[0238]
(R)-9-[2-eicosanoyloxymethyl-4-(t-butyldiphenylsilyl)butyl]guanine,
and
[0239]
(R)-9-[2-docosanoyloxymethyl-4-(t-butyldiphenylsilyl)butyl]guanine.
[0240] An alternative process for the preparation of compounds of
the invention of the formula I wherein R.sub.3 is --OH is shown in
Scheme C. 89
[0241] Referring to Scheme C, malonate 1 (R.sub.4 and R.sub.5 are
lower alkyl or benzyl or the like) is alkylated by reaction with
from about 0.5 to about 2.0 molar equivalents of acetal 2 (R.sub.6
and R.sub.7 are lower alkyl or benzyl and the like or R.sub.6 and
R.sub.7 taken together are --CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2-- or
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2-- and X.sub.1 is a leaving group
(for example, Cl, Br or I, or a sulfonate such as methanesulfonate,
triflate, p-toluenesulfonate, benzenesulfonate and the like)) in
the presence of from about 0.5 to about 2.0 molar equivalents of a
base (for example, potassium t-butoxide or sodium ethoxide or NaH
or KH and the like) in an inert solvent (for example, DMF or THF or
dioxane or dioxolane or N-methylpyrrolidone and the like) at a
temperature of from about -40.degree. C. to about 190.degree. C. to
provide alkylated malonate 3. Alkylated malonate 3 can be purified
by distillation or by first treating the crude alkylated malonate
with dilute aqueous base (for example, 7% aqueous KOH), followed by
removal of volatile impurities by distillation.
[0242] Reduction of 3 with from about 0.5 to about 4.0 molar
equivalents of an ester to alcohol reducing agent (for example,
LiBH.sub.4 or Ca(BH.sub.4).sub.2 or NaBH.sub.4 or LiAlH.sub.4 and
the like) in an inert solvent (for example, THF or methyl t-butyl
ether or t-BuOH and the like) at a temperature of from about
-20.degree. C. to about 100.degree. C. provides diol 4. Enzymatic
esterification of 4 by reaction with from about 1.0 to about 20.0
molar equivalents of a vinyl ester 5 (R.sub.8 is C.sub.1-C.sub.21
saturated or monounsaturated, optionally substituted alkyl) in the
presence of a lipase (for example, Lipase PS-30 or Lipase PPL or
Lipase CCL and the like) or a phospholipase (for example
phospholipase D and the like) provides the desired stereoisomer of
ester 6. This reaction can be carried out in the absence of solvent
or in the presence of an inert solvent (for example, methyl t-butyl
ether or toluene or hexane and the like). The reaction is carried
out at a temperature of from about -20.degree. C. to about
80.degree. C.
[0243] The alcohol substituent of 6 is converted to a leaving group
(for example, a halogen or a sulfonate) by reaction with a
halogenating agent (for example NBS/P(Ph).sub.3 or NCS/P(Ph).sub.3
or POCl.sub.3 or NCS/P(Ph).sub.3/NaI in acetone and like) in an
inert solvent (for example, methylene chloride or toluene or
ethylacetate and the like) or by reaction with from about 0.8 molar
equivalents to about 2.0 molar equivalents of a sulfonyl halide
(for example, benzenesulfonylchloride, toluenesulfonylchloride or
methane sulfonylchloride and the like) in the presence of from
about 1.0 to about 4.0 molar equivalents of a base (for example,
triethylamine or potassium carbonate or pyridine or
dimethylaminopyridine or ethyldiisopropylamine and the like) in an
inert solvent (for example methylene chloride or toluene or
ethylacetate or pyridine or methyl t-butyl ether and the like) at a
temperature of from about -25.degree. C. to about 100.degree. C. to
provide ester 7 (X.sub.2 is a halogen or sulfonate leaving
group).
[0244] Reaction of 7 with from about 0.9 to about 2.0 molar
equivalents of 2-amino-6-chloropurine 8 in the presence of from
about 1.0 to about 6.0 molar equivalents of a base (for example,
potassium carbonate or LiH or NaH or KH or NaOH or KOH or lithium
diisopropylamide or LiN(Si(CH.sub.3).sub.3).sub.2 and the like) in
an inert solvent (for example, DMF or THF or acetonitrile or
N-methylpyrrolidone or ethanol or DMSO and the like) at a
temperature of from about -25.degree. C. to about 140.degree. C.
provides substituted purine 9.
[0245] Alternatively, the base can be a sterically bulky amine base
(for example, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),
1,4-diazabicyclo[2.2.2]octane (Dabco),
1,5-diazabicyclo[4.3.0]non-5-ene (DBN), tetramethylguanidine,
N,N-diisopropylethylamine and the like) or a sterically bulky
phosphazine base (for example, tert-butylimino-tri(pyrro-
lidino)-phosphorane, tert-butylimino-tri(dimethylamino)phosphorane,
tert-octylimino-tri(dimethylamino)phosphorane and the like) in an
inert solvent (for example, THF or DMF or DMSO and the like).
[0246] Alternatively Mitsunobu coupling (for example
P(Ph).sub.3/diethyl azidocarboxylate) of alcohol 6 with
2-amino-6-chloropurine 8 provides 9.
[0247] Reaction of 9 with from about 2.0 to about 20 molar
equivalents of an alcohol R.sub.9OH (R.sub.9 is an alcohol
protecting group such as benzyl or diphenylmethyl and the like) in
the presence of from about 1.0 to about 6.0 molar equivalents of a
base (for example, potassium t-butoxide or potassium carbonate or
NaH or KH or lithium diisopropylamide and the like) in an inert
solvent (for example, THF or DMF and the like) at a temperature of
from about -25.degree. C. to about 150.degree. C. provides alcohol
10.
[0248] Removal of the alcohol protecting group R.sub.9 of 10 (for
example, by catalytic hydrogenation in an inert solvent such as
ethanol or benzyl alcohol or methanol or THF and the like in the
presence of an hydrogenation catalyst such as Pd/C or Pd(OH).sub.2
and the like) provides substituted guanine 11.
[0249] Esterification of 11 by reaction with a) from about 0.8 to
about 2.0 molar equivalents of R.sub.10COOH and a coupling agent
(for example DCC/DMAP) and the like in an inert solvent (for
example THF or DMF and the like) or b) from about 0.8 to about 2.0
molar equivalents of an activated derivative of R.sub.10COOH (for
example, the acid chloride or N-hydroxysuccinimide ester or
R.sub.10C(O)OS(O).sub.2R.sub.30 (R.sub.30 is loweralkyl, phenyl or
toluyl) or R.sub.10C(O)OC(O)R.sub.10 or R.sub.10C(O)OC(O)R.sub.10a
(R.sub.10a is loweralkyl and the like) in the presence of from
about 0 to about 3.0 molar equivalents of a base (for example,
pyridine or dimethylaminopyridine or triethylamine or
ethyldiisopropylamine or N-methylmorpholine or DBU or potassium
carbonate and the like) in an inert solvent (for example, methylene
chloride or THF or pyridine or acetonitrile or DMF and the like) at
a temperature of from about -25.degree. C. to about 100.degree. C.
provides ester 12. R.sub.10 is C.sub.3-C.sub.21 saturated or
monounsaturated, optionally substituted alkyl.
[0250] The acetal substituent of 12 is deprotected and the
resulting aldehyde is reduced by first reacting 12 with from about
0.1 to about 10.0 molar equivalents of an acid (for example,
triflic acid or HCl or formic acid or acetic acid/formic acid or
sulfuric acid and the like) in an inert solvent (for example,
THF/H.sub.2O or methylene chloride/H.sub.2O or
ethylacetate/H.sub.2O or ethanol/H.sub.2O or methanol/H.sub.2O or
water and the like) at a temperature of from about -25.degree. C.
to about 100.degree. C. To the crude reaction mixture is added from
about 0.1 to about 10.0 molar equivalents of a base (for example,
sodium bicarbonate or potassium carbonate or triethylamine or
pyridine or KOH and the like), (optionally, additional inert
solvent (for example, THF and or methylene chloride or ethylacetate
or methyl t-butyl ether or isopropoanol and the like) is added) and
from about 0.3 to about 5.0 molar equivalents of an aldehyde
reducing agent (for example, sodium borohydride or RaNi/H.sub.2 or
borane t-butylamine complex and the like) at a temperature of from
about -25.degree. C. to about 100.degree. C. to provide alcohol 13.
The optical purity of compound 13 can be enhanced by reaction with
optically active oraganic sulfonic acids such as
(S)-(+)-camphorsulfonic acid and the like. A preferred sulfonic
acid for this purpose is (S)-(+)-camphorsulfonic acid.
[0251] Alternatively, the acetal substituent of 12 can be
hydrolyzed by reaction in an inert solvent with an acid resin (for
example, Amberlyst 15 resin, Nafion NR50 resin, Dowex 50WX4-200R
resin or Amerlite 120 resin and the like) to provide the
corresponding aldehyde. The aldehyde can be isolated prior to
reduction to the alcohol 13 as described above or the crude
aldehyde can be reduced directly in situ.
[0252] Reaction of 13 with from about 0.8 to about 3.0 molar
equivalents of N-protected amino acid P.sub.1NHCH(R.sub.11)COOH or
an activated derivative thereof (P.sub.1 is an N-protecting group
(for example, benzyloxycarbonyl, t-butyloxycarbonyl,
allyloxycarbonyl and the like) and R.sub.11 is isopropyl or
isobutyl) in an inert solvent (for example, THF or dioxane or
dioxolane or DMF or methylene chloride and the like) at a
temperature of from about 25.degree. C. to about 100.degree. C.
provides alcohol 14.
[0253] N-deprotection of 14 provides the compound of the invention
of formula I wherein R.sub.3 is --OH. For example, when the
protecting group can be removed by hydrogenation, such as when the
protecting group is Cbz, hydrogenation in the presence of Pd/C in
ethanol or Pd/BaCO.sub.3 or Pd/BaSO.sub.4 and the like in THF or
isopropanol/THF and the like is preferred.
[0254] Alternatively, compound 13 can be reacted with the
symmetrical anhydride derived from P.sub.1NHCH(R.sub.11)COOH (i.e.,
P.sub.1NHCH(R.sub.11)C(O)O--C(O)CH(R.sub.11)NHP.sub.1) to provide
14. The anhydride can be prepared in situ or can be separately
prepared prior to reaction with 13.
[0255] Alternatively, 11 can be prepared by hydrolysis of the ester
of 9 to an alcohol (for example, by reaction with a base such as
K.sub.2CO.sub.3, Li.sub.2CO.sub.3, Na.sub.2CO.sub.3, KHCO.sub.3,
LiOH, NaOH or KOH and the like in an inert solvent such as
methanol, ethanol, isopropanol, THF, water or mixtures thereof and
the like, most prefereably with K.sub.2CO.sub.3 in MeOH/H.sub.2O
and the like), followed by direct conversion of the chloro group to
an --OH (for example, by reaction with an inorganic base such as
KOH or NaOH and the like in H.sub.2O with heating and the
like).
[0256] In another alternative method, 11 can be prepared directly
by hydrolysis of the chloro-ester 9 (for example, by reaction with
an inorganic base such as KOH or NaOH and the like in H.sub.2O with
heating and the like).
[0257] In another alternative, the ester of 9 can be hydrolyzed by
an esterase in water or an aqueous buffer, with or without the
presence of an added organic solvent such as an alcohol (for
example, ethanol or isopropanol and the like), THF, DMF or DMSO and
the like.
[0258] In another alternative method, 11 can be prepared from 9 (or
from the hydroxy compound resulting from the hydrolysis of the
ester in 9) by reaction with an inorganic base (for example, NaOH,
LiOH, KOH and the like, preferably, NaOH) and trimethylamine in an
aqueous solvent.
[0259] In yet another alternative method, 11 can be prepared
directly by hydrolysis of the chloro-ester 9 (for example, by
reaction with 1-3 equivalents of a base such as sodium methoxide
(and the like) in the presence of mercaptoethanol in a mixed
solvent of water and methanol or dioxane (and the like) at a
temperature of from about 20.degree. C. to about relfux and the
like).
[0260] In yet another alternative method, prior to conversion of 9
to 10 or 11, the ester of 9 can be hydrolyzed to the alcohol as
described above. The alcohol can then be reesterified and purified
(for example, from methyl t-butyl ether and the like). This process
leads to an increase in the enantiomeric excess (i.e., purity) of
the resulting ester 9. Preferably, the alcohol is reesterified to
provide the acetate, which is purified from methyl t-butyl
ether.
[0261] In yet another alternative method, 13 can be prepared by
reaction of 9 (wherein R.sub.8=R.sub.10) with formic acid,
optionally with heating, followed by reduction of the aldehyde to
give 13.
[0262] In yet another alternative, 13 can be prepared from 11
without isolation of intermediates and with in situ generation of
the esterification agent, thus increasing purity of the resulting
product and allowing increased throughput in the process.
[0263] Another alternative process for the preparation of compounds
of Formula I wherein R.sub.3 is --OH is shown in Scheme D. 1011
[0264] Malonate 1 (R.sub.4 and R.sub.5 are lower alkyl or benzyl
and the like) is alkylated with from about 0.5 to about 2.0 molar
equivalents of ether 15 wherein X, is a leaving group (for example
Cl, Br or I, or a sulfonate such as methane sulfonate, triflate,
p-toluenesulfonate, benzenesulfonate and the like) and R.sub.12 is
--CH(Ph).sub.2, --C(Ph).sub.3 or --Si(t-Bu)(Me).sub.2 and the like
(Ph=phenyl) in the presence of from about 0.5 to about 2.0 molar
equivalents of a base (for example potassium t-butoxide or sodium
ethoxide or NaH or KH and the like) in an inert solvent (for
example DMF or THF or dioxane or dioxolane or N-methylpyrrolidinone
and the like) at a temperature of from about -40.degree. C. to
about 190.degree. C. to provide alkylated malonate 16.
[0265] Reduction of 16 with from about 0.5 to about 4.0 molar
equivalents of an ester to alcohol reducing agent (for example
LiBH.sub.4 or Ca(BH.sub.4).sub.2 or NaBH.sub.4 or LiAlH.sub.4 and
the like) in an inert solvent (for example, THF or methyl t-butyl
ether or ethanol or t-butanol and the like) at a temperature of
from about -20.degree. C. to about 100.degree. C. provides diol 17.
Enzymatic esterification of 17 by reaction with from about 1.0 to
about 20.0 molar equivalents of a vinyl ester 5 (R.sub.8 is
C.sub.1-C.sub.21 saturated or monounsaturated, optionally
substituted alkyl) in the presence of a lipase (for example, Lipase
PS-30 or Lipase PPL or Lipase CCL and the like) or a phospholipase
(for example phospholipase D and the like) provides the desired
stereoisomer of ester 18. The reaction can be carried out in the
absence of solvent or in the presence of an inert solvent (for
example methyl t-butyl ether or toluene or hexane or the like). The
reaction is carried out at a temperature of from about -20.degree.
C. to about 80.degree. C.
[0266] The alcohol substituent of 18 is converted to a leaving
group (for example a halogen or sulfonate) by reaction with a
halogenating agent (for example NBS/P(Ph).sub.3 or NCS/P(Ph).sub.3
or POCl.sub.3 or NCS/P(Ph).sub.3/NaI in acetone and the like) in an
inert solvent (for example methylene chloride or toluene or
ethylacetate and the like) or by reaction with from about 0.8 molar
equivalents to about 2.0 molar equivalents of a sulfonyl halide
(for example benzenesulfonylchloride, toluenesulfonylchloride or
methane sulfonylchloride and the like) in the presence of from
about 1.0 to about 4.0 molar equivalents of a base (for example
triethylamine or potassium carbonate or pyridine and the like) in
an inert solvent (for example, methylene chloride or toluene or
ethyl acetate or methyl t-butyl ether and the like) at a
temperature of from about -25.degree. C. to about 100.degree. C. to
provide ester 19 (X.sub.2 is a halogen or sulfonate leaving
group).
[0267] Reaction of 19 with from about 0.9 to about 2.0 molar
equivalents of 2-amino-4-chloropurine 8 in the presence of from
about 1.0 to about 6.0 molar equivalents of a base (for example
potassium carbonate or LiH or NaH or KH or NaOH or KOH or lithium
diisopropylamide or LiN(Si(CH.sub.3).sub.3).sub.2 and the like) in
an inert solvent (for example DMF or THF or acetonitrile or
N-methylpyrrolidone or ethanol and the like) at a temperature of
from about -25.degree. C. to about 140.degree. C. provides
substituted purine 20.
[0268] Alternatively, Mitsunobu coupling (for example,
P(PH).sub.3/diethyl azidocarboxylate) of alcohol 18 with
2-amino-4-chloropurine 8 provides 20.
[0269] Reaction of 20 with from about 2.0 to about 20.0 molar
equivalents of an alcohol R.sub.9OH(R.sub.9 is an alcohol
protecting group such as benzyl or diphenylmethyl and the like) in
the presence of from about 1.0 to about 6.0 molar equivalents of a
base (for example, potassium t-butoxide or potassium carbonate or
NaH or KH or lithium diisopropylamide and the like in an inert
solvent (for example, THF or DMF and the like) at a temperature of
from about -25.degree. C. to about 150.degree. C. provides alcohol
21.
[0270] Removal of the alcohol protecting group R.sub.9 of 21 (for
example by catalytic hydrogenation in an inert solvent such as
ethanol or benzyl alcohol or methanol or THF and the like in the
presence of an hydrogenation catalyst such as Pd/C or Pd(OH).sub.2
and the like) provides substituted guanine 22, which can be
esterified as described in Scheme C (i.e., 11 to 12) to provide
23.
[0271] The ether substitutent of 23 is deprotected by reaction with
a) a reducing agent (for example, HCO.sub.2H and Pd/C and the like)
wherein R.sub.12 is --CH(Ph).sub.2 or --C(Ph).sub.3, or b) a
desilylating agent (for example Bu.sub.4NF and the like) wherein
R.sub.12 is --Si(t-Bu)(Me).sub.2 and the like to provide 13.
[0272] Alcohol 13 can be converted to I as outlined in Scheme
C.
[0273] Alternatively, 22 can be prepared by hydrolysis of the ester
of 20 to an alcohol (for example, by reaction with K.sub.2CO.sub.3
in MeOH/H.sub.2O and the like), followed by direct conversion of
the chloro group to an --OH (for example, by reaction with KOH in
H.sub.2O with heating and the like).
[0274] In another alternative method, 22 can be prepared directly
by hydrolysis of the chloro-ester 20 (for example, by reaction with
KOH in H.sub.2O with heating and the like).
[0275] In another alternative method, 22 can be prepared from 20
(or from the hydroxy compound resulting from the hydrolysis of the
ester in 20) by reaction with an inorganic base (for example, NaOH,
LiOH, KOH and the like, preferably, NaOH) and trimethylamine in an
aqueous solvent.
[0276] In yet another alternative method, 22 can be prepared
directly by hydrolysis of the chloro-ester 20 (for example, by
reaction with 1-3 equivalents of a base such as sodium methoxide
(and the like) in the presence of mercaptoethanol in a mixed
solvent of water and methanol or dioxane (and the like) at a
temperature of from about 20.degree. C. to about relfux and the
like).
[0277] In yet another alternative method, 23 can be prepared by
reaction of 20 (wherein R.sub.8=R.sub.10) with formic acid,
optionally with heating, followed by reduction of the aldehyde to
give 23.
[0278] An additional alternative involves enzymatic esterification
of alcohol 4 or 17 with the vinyl ester
CH.sub.2.dbd.CH--OC(O)R.sub.10 (i.e., R.sub.8=R.sub.10 in Schemes C
and D) to directly incorporate into 6 or 18 the desired carboxylic
acid ester of the final product I. This allows the elimination of
the ester hydrolysis and reesterification involved in going from 9
to 12 or from 20 to 23.
[0279] The processes of Schemes C and D are characterized by the
fact that each of the hydroxyl groups of the acyclic side chain is
differentiated by the use of different hydroxy protecting groups or
precursor groups. This allows the selective acylation of each of
the hydroxy groups with either an amino acid or a fatty acid
group.
[0280] Schemes C and D have been illustrated and described with
reference to embodiments of the invention wherein R.sub.1 is
derived from an amino acid and R.sub.2 is derived from a fatty
acid. However, it will be apparent that respective converse schemes
will apply to compounds where R.sub.1 is derived from a fatty acid
and R.sub.2 is derived from an amino acid. 12
[0281] Yet another method for preparing compounds of Formula I is
shown in Scheme E. Enzymatic esterification of 4 (see Scheme C) by
reaction with from about 1.0 to about 20.0 molar equivalents of a
vinyl ester 24 (R.sub.10 is C.sub.3-C.sub.21 saturated or
monounsaturated, optionally substituted alkyl) in the presence of a
lipase (for example, Lipase PS-30 or Lipase PPL or Lipase CCL and
the like) or a phospholipase (for example phospholipase D and the
like) provides the desired stereoisomer of ester 25. This reaction
can be carried out in the absence of solvent or in the presence of
an inert solvent (for example, methyl t-butyl ether or toluene or
hexane and the like). The reaction is carried out at a temperature
of from about -20.degree. C. to about 80.degree. C.
[0282] The alcohol substituent of 25 is converted to a leaving
group (for example, a halogen or a sulfonate) by reaction with a
halogenating agent (for example NBS/P(Ph).sub.3 or NCS/P(Ph).sub.3
or POCl.sub.3 or NCS/P(Ph).sub.3/NaI in acetone and like) in an
inert solvent (for example, methylene chloride or toluene or
ethylacetate and the like) or by reaction with from about 0.8 molar
equivalents to about 2.0 molar equivalents of a sulfonyl halide
(for example, benzenesulfonylchloride, toluenesulfonylchloride or
methane sulfonylchloride and the like) in the presence of from
about 1.0 to about 4.0 molar equivalents of a base (for example,
triethylamine or potassium carbonate or pyridine or
dimethylaminopyridine or ethyldiisopropylamine and the like) in an
inert solvent (for example methylene chloride or toluene or
ethylacetate or pyridine or methyl t-butyl ether and the like) at a
temperature of from about -25.degree. C. to about 100.degree. C. to
provide ester 26 (X.sub.2 is a halogen or sulfonate leaving
group).
[0283] The acetal substituent of 26 is hydrolyzed to the aldehyde
27 by reacting 26 with an acid (for example, trifluoroacetic acid,
triflic acid or HCl or formic acid or acetic acid/formic acid or
sulfuric acid and the like) in an inert solvent (for example,
THF/H.sub.2O or methylene chloride/H.sub.2O or
ethylacetate/H.sub.2O or ethanol/H.sub.2O or methanol/H.sub.2O or
water and the like) at a temperature of from about -25.degree. C.
to about 100.degree. C.
[0284] To the aldehyde 27 in an inert solvent (for example, THF and
or methylene chloride or ethylacetate or methyl t-butyl ether or
isopropoanol and the like) is added an aldehyde to alcohol reducing
agent (for example, sodium borohydride or RaNi/H.sub.2 or borane
t-butylamine complex and the like) at a temperature of from about
-25.degree. C. to about 100.degree. C. to provide the corresponding
alcohol.
[0285] Reaction of the resulting alcohol with from about 0.8 to
about 3.0 molar equivalents of N-protected amino acid
P.sub.1NHCH(R.sub.11)COOH or an activated derivative thereof
(P.sub.1 is an N-protecting group (for example, benzyloxycarbonyl,
t-butyloxycarbonyl, allyloxycarbonyl, trichloroethylcarbonyl and
the like) and R.sub.11 is isopropyl or isobutyl) in an inert
solvent (for example, THF or dioxane or dioxolane or DMF or
methylene chloride and the like) at a temperature of from about
25.degree. C. to about 100.degree. C. provides diester 28.
[0286] Alternatively the alcohol can be reacted with the
symmetrical anhydride derived from P.sub.1NHCH(R.sub.11)COOH (i.e.,
P.sub.1NHCH(R.sub.11)C(O)O--C(O)CH(R.sub.11)NHP.sub.1) to provide
28.
[0287] Conversion of 27 to 28 can be accomplished with or without
isolation/purification of the intermediate alcohol. A preferred
aldehyde to alcohol reducing agent is borane t-butylamine complex.
A preferred esterification agent is the symmetrical anhydride.
[0288] Reaction of 28 with purine 29 in the presence of a base (for
example potassium carbonate or LiH or NaH or KH or NaOH or KOH or
lithium diisopropylamide or LiN(Si(CH.sub.3).sub.3).sub.2 and the
like) in an inert solvent (for example, DMF and the like) provides
30. Purine 29 is prepared from 6-chloro-2-amino purine by reaction
with R.sub.9OH in an inert solvent (for example, toluene or THF and
the like) in the presence of a base (for example, NaH or KH or NaOH
or KOH or potassium t-butoxide and the like). A preferred process
for the the preparation of purine 29 involves reaction of
2-amino-6-chloropurine with neat R.sub.9-OH in the presence of a
base such as NaOH or KOH or potassium t-butoxide and the like.
Substituted purine 30 is deprotected to provide the compound of
Formula I.
[0289] Alternatively, in the reaction of 28 with 29, the base can
be a sterically bulky amine base (for example,
1,8-diazabicyclo[5.4.0]-undec-7- -ene (DBU),
1,4-diazabicyclo[2.2.2]octane (Dabco), 1,5-diazabicyclo[4.3.0]-
non-5-ene (DBN), tetramethylguanidine, N,N-diisopropylethylamine
and the like) or a sterically bulky phosphazine base (for example,
tert-butylimino-tri(pyrrolidino)-phosphorane,
tert-butylimino-tri(dimethy- lamino)phosphorane,
tert-octylimino-tri(dimethylamino)phosphorane and the like) in an
inert solvent (for example, THF or DMF or DMSO and the like).
13
[0290] Yet another method for preparing compounds of Formula I is
shown in Scheme F. Reaction of 28 with amino-chloropurine 8 in the
presence of a base (for example potassium carbonate or LiH or NaH
or KH or NaOH or KOH or lithium diisopropylamide or
LiN(Si(CH.sub.3).sub.3).sub.2 and the like) in an inert solvent
(for example, DMF THF and the like) provides 31. Hydrolysis of 31
to 14 can be accomplished under basic or acidic conditions (for
example, with trimethlyamine or DABCO or KOH or LiOH or NaOH and
the like in water/THF or methylene chloride and the like or with
acetic acid and the like).
[0291] Alternatively, 8 can be be alkylated with 28 using a
sterically bulky amine base (for example,
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),
1,4-diazabicyclo[2.2.2]octane (Dabco),
1,5-diazabicyclo[4.3.0]non-5-ene (DBN), tetramethylguanidine,
N,N-diisopropylethylamine and the like) or a sterically bulky
phosphazine base (for example, tert-butylimino-tri(pyrro-
lidino)-phosphorane, tert-butylimino-tri(dimethylamino)phosphorane,
tert-octylimino-tri(dimethylamino)phosphorane and the like) in an
inert solvent (for example, THF or DMF or DMSO and the like).
[0292] In each of Schemes C, D and F, the 2-amino-6-chloro-purine
(8) can be replaced with 2-amino-6-iodo-purine or
2-amino-6-bromopurine, which can be alkylated and then transformed
to the substituted guanine in a manner analogous to that disclosed
for alkylation and transformation of 8. 14
[0293] Yet another method for preparing the compounds of formula I
is shown in Scheme G. Alkylation of 32 with 7 in the presence of a
base (for example, potassium carbonate, LiH, NaH and the like) in
an inert solvent (for example, DMF THF and the like) provides 33.
R.sub.25 is hydrogen or --C(O)NR.sub.27R.sub.28 wherein R.sub.27
and R.sub.28 are independently selected from loweralkyl, phenyl and
benzyl or R.sub.27 and R.sub.28, taken together with the nitrogen
to which they are attached, form a pyrrolidinyl group or a
piperidinyl group. R.sub.26 is loweralkyl, phenyl or benzyl.
[0294] Hydrolysis of 33 to 11 can be accomplished under basic
conditions (for example, with KOH in water and the like).
[0295] Alternatively, 32 can be alkylated with 7 using a sterically
bulky amine base (for example, 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU), 1,4-diazabicyclo[2.2.2]octane (Dabco),
1,5-diazabicyclo[4.3.0]non-5-ene (DBN), tetramethylguanidine,
N,N-diisopropylethylamine and the like) or a sterically bulky
phosphazine base (for example, tert-butylimino-tri(pyrro-
lidino)-phosphorane, tert-butylimino-tri(dimethylamino)phosphorane,
tert-octylimino-tri(dimethylamino)phosphorane and the like) in an
inert solvent (for example, THF or DMF or DMSO and the like).
DETAILED DESCRIPTION OF THE INVENTION
[0296] The invention will now be illustrated by way of example only
with reference to the following non-limiting Examples, comparative
examples and the accompanying Figures, in which:
[0297] FIG. 1 depicts plasma H2G levels as a function of time in
cynomolgus monkeys administered with a compound of the invention or
with an alternative prodrug derivative of H2G, as further explained
in Biological Example 3; and
[0298] FIG. 2 depicts survival as a function of time for Herpes
simplex infected mice administered with various doses of a compound
of the invention or a prior art antiviral, as further explained in
Biological Example 4.
EXAMPLE 1
(R)-9-[2-(Stearoyloxymethyl)-4-(L-valyloxy)butyl]guanine
[0299] This example illustrates the application of preparation
scheme A.
a) (R)-9-[4-(N-tert-Butoxycarbonyl-L-valyloxy)-2-(hydroxymethyl)
butyl]guanine
[0300] H2G (5 g, 19.7 mmol) was dissolved in DMF (300 ml) under
heating and was cooled to room temperature before addition of
N-t-Boc-L-valine (5.58 g, 25.7 mmol), DMAP (0.314 g, 2.57 mmol) and
DCC (6.52 g, 31.6 mmol). The mixture was stirred at room
temperature for 24 h and was then filtered. The product was
chromatographed on silica gel and eluted with CH.sub.2Cl.sub.2/MeOH
to give 2.4 g of the desired intermediate product.
[0301] .sup.1H-NMR (250 MHz, DMSO-d.sub.6): .delta. 0.95 (d, 6H),
1.47 (s, 9H), 1.5-1.8 (m, 2H), 1.96-2.20 (m, 2H), 3.40 (m, 2H),
3.91 (t, 1H), 4.05 (m, 2H), 4.21 (t, 2H), 4.89 (t, 1H), 6.6 (br s,
2H), 7.27 (d, 1H), 7.75 (s, 1H), 10.7 (br s, 1H).
b)
(R)-9-[4-(N-tert-Butoxycarbonyl-L-valyloxy)-2-(stearoyloxymethyl)
butyl]guanine
[0302] The product from step a) (185 mg, 0.41 mmol) was dissolved
in pyridine (5 ml), the solution was cooled in an ice bath and
stearoyl chloride (179 .mu.l, 0.531 mmol) was added. The solution
was kept in the ice bath for 2 h, then at room temperature for 1 h.
It was then evaporated and chromatographed on silica gel. It was
eluted with dichloromethane/methanol to give 143 mg of the desired
intermediate product.
c) (R)-9-[2-(Stearoyloxymethyl)-4-(L-valyloxy)butyl]guanine
[0303] The product from step b) (138 mg, 0.192 mmol) was cooled in
an ice bath and trifluoroacetic acid (5 ml) was added. The solution
was kept in the ice bath for 45 minutes and was then evaporated to
give an oil. Water (0.5 to 1 ml) was added and evaporated twice.
The residue was once more dissolved in water (5 ml), filtered and
freeze-dried to give 148 mg of the desired product as the
bistrifluoracetate salt.
[0304] .sup.1H NMR (250 MHz, DMSO-d.sub.6): .delta. 0.97 (t, 3H),
1.05 (dd, 6H), 1.34 (br s, 28H), 1.59 (m, 2H), 1.80 (m, 2H), 2.25
(m, 1H), 2.36 (t, 2H), 2.50 (m, 1H), 3.98-4.18 (m, 5H), 4.35 (t,
2H), 6.6 (br s, 2H), 8.0 (br s, 1H), 8.4 (br s, 3H), 10.9 (br s,
1H).
EXAMPLE 2
(R)-9-[2-(Myristoyloxymethyl)-4-(L-valyloxy)butyl]guanine
[0305] The titled compound was obtained as the bistrifluoracetate
salt in a manner analogous to Example 1 using myristoyl chloride
instead of stearoyl chloride in step b).
[0306] .sup.1H NMR (250 MHz, DMSO-d.sub.6): .delta. 0.97 (t, 3H),
1.05 (dd, 6H), 1.34 (br s, 20H), 1.57 (m, 2H), 1.78 (m, 2H), 2.24
(m, 1H), 2.35 (t, 2H), 2.51 (m, 1H), 3.97-4.20 (m, 5H), 4.36 (t,
2H), 6.8 (br s, 2H), 8.2 (br s, 1H), 8.5 (br s, 3H), 11.1 (br s,
1H).
EXAMPLE 3
(R)-9-[2-(Oleoyloxymethyl)-4-(L-valyloxy)butyl]guanine
[0307] The titled compound was obtained as the bistrifluoroacetyl
salt in a manner analogous to Example 1 using oleoyl chloride
instead of stearoyl chloride in step b).
[0308] .sup.1H NMR (250 MHz, DMSO-d.sub.6): .delta. 0.96 (t, 3H),
1.05 (dd, 6H), 1.35 (br s, 20H), 1.59 (m, 2H), 1.76 (m, 2H), 2.09
(m, 4H), 2.24 (m, 1H), 2.35 (t, 2H), 2.50 (m, 1H), 3.97-4.17 (m,
5H), 4.35 (t, 2H), 5.43 (t, 2H), 6.7 (br s, 2H), 8.0 (br s, 1H),
8.5 (br s, 3H), 11.1 (br s, 1H).
EXAMPLE 4
(R)-9-[2-(Butyryloxymethyl)-4-(L-valyloxy)butyl]guanine
a) (R)-9-[4-(N-tert-Butoxycarbonyl-L-valyloxy)-2-(butyryloxymethyl)
butyl]guanine
[0309] DCC (110 mg, 0.53 mmol) was dissolved in dichloromethane (10
ml) and butyric acid (82 mg, 0.93 mmol) was added. After 4 hours at
room temperature the mixture was filtered and the filtrate was
evaporated. The residue was dissolved in pyridine (5 ml) and
(R)-9-[4-(N-tert-Butoxycarbo-
nyl-L-valyloxy)-2-hydroxymethylbutyl]guanine (200 mg, 0.44 mmol)
(Example 1, step a) was added. The mixture was stirred for 120
hours at room temperature. According to TLC the reaction was
incomplete and more anhydride was made using the procedure above.
This anhydride was added and the mixture was stirred for an
additional 20 hours. The reaction mixture was evaporated and
chromatographed first on silica gel and then on aluminium oxide, in
both cases eluted with dichloromethane/methanol to give 79 mg of
the intermediate product.
b) (R)-9-[2-(Butyryloxymethyl)-4-(L-valyloxy)butyl]guanine
[0310] The intermediate product of step a was deprotected in a
manner analogous to Example 1, step c to give 84 mg of the desired
product as the bistrifluoracetate salt.
[0311] .sup.1H NMR (250 MHz, D.sub.2O): .delta. 0.88 (t, 3H), 1.06
(dd, 6H), 1.53 (m, 2H), 1.93 (q, 2H), 2.25 (t, 2H), 2.36 (m, 1H),
2.60 (m, 1H), 4.06 (d, 1H), 4.14-4.30 (m, 2H), 4.43 (m, 4H), 8.99
(br s, 1H).
EXAMPLE 5
(R)-9-[2-(Decanoyloxymethyl)-4-(L-valyloxy)butyl]guanine
[0312] The titled compound was obtained as the bistrifluoroacetate
salt in a manner analogous to Example 1 using decanoyl chloride
instead of stearoyl chloride in step b.
[0313] .sup.1H NMR (250 MHz, D.sub.2O): (0.90 (m, 3H), 1.01 (d,
6H), 1.28 (br s, 12H), 1.5 (m, 2H), 1.8 (m, 2H), 2.3 (m, 3H), 2.5
(m, 1H), 4.04.4 (m, 7H), 8.1 (br s, 1H).
EXAMPLE 6
(R)-9-[2-Docosanoyloxymethyl-4-(L-valyloxy)butyl]guanine
[0314] The titled compound was obtained as the bistrifluoroacetate
salt in a manner analogous to Example 1 but using in step b the
DMAP/DCC conditions of Example 1, step a) in conjunction with
docosanoic acid in place of the stearoyl chloride and a mixture of
DMF and dichloromethane as solvent.
[0315] .sup.1H NMR (250 MHz, DMSO-d.sub.6): .delta. 0.97 (t, 3H),
1.05 (dd, 6H), 1.34 (br s, 36. H), 1.58 (m, 2H), 1.77 (m, 2H), 2.24
(m, 1H), 2.35 (t, 2H), 2.50 (m, 1H), 3.97-4.17 (m, 5H), 4.35 (t,
2H), 6.7 (br s, 2H), 8.1 (br s, 1H), 8.4 (br s, 3H), 11.0 (br s,
1H).
EXAMPLE 7
R-9-(4-(L-Isoleucyloxy)-2-(stearoyloxymethyl)butyl(guanine
[0316] This example illustrates the application of preparative
scheme B.
a) (R)-9-[2-hydroxymethyl
4-(t-butyldiphenylsilyloxy)butyl]guanine
[0317] H2G (2 g, 8 mmole) was coevaporated with dry DMF two times
and was then suspended in dry DMF (120 ml) and pyridine (1 ml). To
the suspension was added dropwise t-butyldiphenylchlorosilane (2.1
ml, 8.2 mmole) in dichloromethane (20 ml) at 0 (C over a period of
30 min. The reaction mixture became a clear solution at the
completion of the dropwise addition. The reaction continued at
0.degree. C. for two hours and was then kept at 4.degree. C.
overnight. Methanol (5 ml) was added to the reaction. After 20 min
at room temperature, the reaction mixture was evaporated to a small
volume, poured into aqueous sodium hydrogen carbonate solution and
extracted with dichloromethane two times. The organic phase was
dried over sodium sulphate and evaporated in vacuo. The product was
isolated by silica gel column chromatography using a
methanol/dichloromethane system with a stepwise increasing MeOH
concentration. The product was eluted with 7% MeOH in
CH.sub.2Cl.sub.2 to yield 1.89 g.
b)
(R)-9-[2-(Stearoyloxymethyl)-4-(t-butyldiphenylsilyloxy)butyl]guanine
[0318] (R)-9-[2-Hydroxymethyl
4-(t-butyldiphenylsilyloxy)butyl]guanine (2.31 g, 5 mmole) was
coevaporated with dry pyridine twice and dissolved in pyridine (20
ml). To the solution was slowly added dropwise stearoyl chloride
(1.86 ml, 5.5 mmole, technical grade) in dichloromethane (2 ml) at
-5.degree. C. The reaction was kept at the same temperature for 1
hr and then at 5.degree. C. for 2 hr. The reaction was monitored by
TLC. Additional stearoyl chloride (0.29 ml) at -5.degree. C. was
added due to incompletion of reaction. After 30 min at 5.degree.
C., methanol (3 ml) was added and the reaction mixture stirred for
20 min. It was then poured into aqueous sodium hydrogen carbonate
solution, and extracted with dichloromethane. The organic phase was
dried and the product purified by silica gel column chromatography
with stepwise increasing MeOH, eluting with 3.5% MeOH in
CH.sub.2Cl.sub.2. (Yield 2.7 g).
c) (R)-9-[(4-Hydroxy-2-(stearoyloxymethyl)butyl]guanine
[0319]
(R)-9-[2-(Stearoyloxymethyl)-4-(t-butyldiphenylsilyloxy)butyl]guani-
ne (2.7 g, 3.56 mmole) was dissolved in dry THF (30 ml) and
hydrogen fluoride-pyridine (1.5 ml) added to the solution. The
reaction was kept at 4.degree. C. overnight and monitored by TLC.
The reaction reached about 80% conversion. Additional HF-pyridine
was added (0.75 ml). After 4 hr, TLC showed that the starting
material had disappeared. The reaction mixture was concentrated in
vacuo without raising the temperature and more pyridine (5 ml) was
added and evaporated again. The product was isolated by silica gel
column chromatography. (Yield 1.26 g).
d)
(R)-9-[4-(N-BOC-L-isoleucyloxy)-2-(stearoyloxymethyl)butyl]guanine
[0320] (R)-9-(4-Hydroxy-2-(stearoyloxymethyl)butyl(guanine (135 mg,
0.26 mmole) and N-BOC-L-isoleucine (180 mg, 0.78 mmole) were
coevaporated with dry DMF twice and dissolved in the same solvent
(3.5 ml). To the solution was added 1,3-dicyclohexylcarbodiimide
(160 mg, 0.78 mmole) and 4-dimethylaminopyridine (4.8 mg, 0.039
mmole). After reaction for 18 hours, the reaction mixture was
filtered through Celite and worked up in a conventional manner. The
product was isolated by silica gel column chromatography, eluting
at 5% MeOH in CH.sub.2Cl.sub.2. (Yield 160 mg)
e)
(R)-9-[4-(L-Isoleucyloxy)-2-(stearoyloxymethyl)-butyl]guanine
[0321]
(R)-9-[4-(N-BOC-L-isoleucyloxy)-2-(stearoyloxymethyl)butyl]guanine
(150 mg, 0.205 mmole) from step d) was treated with trifluoroacetic
acid (3 ml) at 0.degree. C. for 20 min. The solution was evaporated
in vacuo. The residue was coevaporated with toluene twice and kept
under vacuum for several hours. The residue was dissolved in MeOH
(2 ml) and evaporated to give the trifluoracetate salt as a
glass-like product. (Yield 191 mg).
[0322] .sup.1H NMR (DMSO-d.sub.6+D.sub.2O): .delta. 8.35 (s, 1H,
base), 4.21 (t, 2H, H-4), 4.10 (d, 2H) 3.96 (d, 2H), 3.90 (d, 1H,
isoleucine), 2.48 (m, 1H, H-2), 2.15 (2H, stearoyl), 1.85 (m, 1H,
isoleucine), 1.68 (m, 2H), 1.48 (m, 4H), 1.68 (m, 28H), 0.81 (m,
9H).
EXAMPLE 8
(R)-9-[2-(Decanoyloxymethyl)-4-(L-isoleucyloxy)butyl]guanine
[0323] The title compound was obtained as the bistrifluoroacetate
salt in a manner analogous to Example 7 using decanoyl chloride
instead of stearoyl chloride in step b).
[0324] .sup.1H NMR (DMSO-d.sub.6): .delta. 11.1 (s, 1H, NH), 8.35
(s, br, 3H), 8.28 (s, 1H, base), 6.75 (s, 2H, NH2), 4.23 (t, 2H),
4.07 (d, 2H), 4.05 (m, 3H), 2.4 (m, 1H), 2.21 (t, 2H), 1.83 (m,
1H), 1.66 (m, 2H), 1.45 (m, 2H), 1.39 (m, 2H), 1.22 (s, 12H), 0.84
(m, 9H).
EXAMPLE 9
(R)-9-[4-(L-Isoleucyloxy)-2-(myristoyloxymethyl)butyl]guanine
[0325] The title compound was obtained as the bistrifluoroacetyl
salt in a manner analogous to Example 1 using N-BOC-L-isoleucine
instead of N-BOC-valine in step a) and myristoyl chloride instead
of stearoyl chloride in step b).
[0326] .sup.1H-NMR (DMSO-d.sub.6): .delta. 10.99 (s, 1H), 8.34 (br
s, 3H) 8.15 (s, 1H), 6.67 (br s, 2H), 4.23 (t, 2H), 4.05 (d, 2H),
3.97 (m, 3H), 2.48 (m, 1H), 2.20 (t, 2H), 1.85 (m, 1H), 1.65 (m,
2H), 1.41 (m, 4H), 1.23 (s, 20H), 0.85 (m, 9H).
EXAMPLE 10
(R)-9-[2-(4-Acetylbutyryloxymethyl-4-(L-valyloxy)butyl]guanine
[0327] The titled compound was obtained as the bistrifluoroacetate
salt in a manner analogous to Example 1 but using in step b) the
DCC/DMAP conditions of Example 1, step a) in conjunction with
4-acetylbutyric acid instead of stearoyl chloride.
[0328] .sup.1H-NMR (250 MHz, DMSO-d.sub.6): 61.05 (dd, 6H), 1.77
(m, 4H), 2.19 (s, 3H), 2.24 (m, 1H), 2.36 (t, 2H), 2.44-2.60 (m,
3H), 3.95-4.20 (m, 5H), 4.36 (m, 2H), 6.8 (br s, 2H), 8.3 (br s,
1H), 8.5 (br s, 3H), 11.1 (br s, 1H).
EXAMPLE 11
(R)-9-[2-Dodecanoyloxymethyl-4-(L-valyloxy)butyl]guanine
[0329] The titled compound was obtained as the bistriflouroacetate
salt in a manner analogous to Example 1 using dodecanoyl chloride
instead of stearoyl chloride in step b).
EXAMPLE 12
(R)-9-[2-Palmitoyloxymethyl-4-(L-valyloxy)butyl]guanine
[0330] The titled compound was obtained as the bistriflouroacetate
salt in a manner analogous to Example 1 using palmitoyl chloride
instead of stearoyl chloride in step b).
[0331] .sup.1H-NMR (250 MHz, DMSO-d.sub.6): .delta. 0.97 (t, 3H),
1.05 (m, 6H), 1.35 (br s, 24H), 1.58 (m, 2H), 1.78 (m, 2H), 2.25
(m, 1H), 2.35 (t, 2H), 2.51 (m, 1H), 3.97-4.18 (m, 5H), 4.35 (t,
2H), 6.7 (br s, 2H), 8.1 (br s, 1H), 8.5 (br s, 3H), 11.0 (br s,
1H).
EXAMPLE 13
(R)-2-Amino-9-(2-stearoyloxymethyl-4-(L-valyloxy)butyl)purine
[0332] This example shows the deoxygenation of group R.sub.1.
a)
(R)-2-Amino-9-(2-stearoyloxymethyl-4-(N-tert-butoxycarbonyl-L-valyloxy)-
butyl)-6-chloropurine
[0333] To a solution of
(R)-9-(2-stearoyloxymethyl-4-(N-tert-butoxycarbony-
l-L-valyloxy)butyl)guanine from step b of Example 1 (646 mg, 0.9
mmole) in acetonitrile were added tetramethylammonium chloride (427
mg, 2.7 mmole), N,N-diethylaniline (0.716 ml, 4.5 mmole) and
phosphorous oxychloride (0.417 ml, 4.5 mmole). The reaction was
kept under reflux and the progression monitored by TLC. After 3
hours the reaction mixture was evaporated in vacuo and the residue
was dissolved in dichloromethane, then poured into cold sodium
hydrogen carbonate aqueous solution. The organic phase was
evaporated and purified by silica gel column chromatography. Yield:
251 mg.
[0334] .sup.1H-NMR (CDCl.sub.3): .delta. 7.76 (1H, H-8), 5.43 (br,
2H, NH2), 4.45-4.00 (m, 7H), 2.53 (m, 1H), 2.28 (t 2H), 2.12 (m,
1H), 1.75 (m, 2H), 1.59 (m, 2H), 1.43 (9H), 1.25 (m, 28H), 0.96 (d,
3H), 0.87 (m, 6H).
b)
(R)-2-Amino-9-(2-stearoyloxmethyl-4-(N-tert-butoxycarbonyl-L-valyloxy)b-
utyl)purine
[0335] To the solution of
(R)-2-amino-9-(2-stearoyloxymethyl-4-(N-tert-but-
oxycarbonyl-L-valyloxy)butyl)-6-chloropurine (240 mg, 0.33 mmole)
in methanol/ethyl acetate (6 ml, 3:1 v/v) were added ammonium
formate (105 mg, 1.65 mmole) and 10% palladium on carbon (15 mg).
The reaction was kept under reflux for 1 hour and recharged with
ammonium formate (70 mg). After one hour more the TLC showed
completion of the reaction and the mixture was filtered through
Celite and washed extensively with ethanol. The filtrate was
evaporated and purified by silica gel column. Yield: 193 mg.
[0336] .sup.1H-NMR (CDCl.sub.3): .delta. 8.69 (s, 1H, H-6), 7.74
(s, 1H, H-8), 5.18 (br, s, 2H, NH2), 4.45-4.01 (m, 7H), 2.55 (m,
1H), 2.28 (t, 2H), 2.10 (m, 1H), 1.75 (m, 2H), 1.60 (m, 2H), 1.43
(s, 9H), 1.25 (s, 28H), 0.96 (d, 3H), 0.87 (m, 6H).
c)
(R)-2-Amino-9-(2-stearoyloxymethyl-4-(L-valyloxy)butyl)purine
[0337]
(R)-2-Amino-9-(2-Stearoyloxmethyl-4-(N-tert-butoxycarbonyl-L-valylo-
xy)butyl)purine (180 mg, 0.26 mmole) was treated with
trifluoroacetic acid (5 ml) at 0.degree. C. for 40 min. It was then
evaporated in vacuo and coevaporated successively with toluene and
methanol. The residue was freeze-dried overnight to give 195 mg of
the desired product.
[0338] .sup.1H-NMR (DMSO-d.sub.6): .delta. 8.78 (s, 1H, H-6), 8.32
(br, 3H), 8.29 (s, 1H, H-8), 4.27 (t, 2H), 4.13 (d, 2H), 3.98 (t,
2H, 2H), 3.89 (m, 1H), 2.47 (m, 1H), 2.18 (m, 3H), 1.43 (m, 2H),
1.23 (28H), 0.93 (m, 6H), 0.85 (t, 3H).
EXAMPLE 14
Alternative preparation of
(R)-9-[4-Hydroxy-2-(stearoyloxymethyl)butyl]gua- nine
a) Preparation of ethyl 4,4-diethoxy-2-ethoxycarbonyl-butyrate
[0339] 15
[0340] Potassium tert-butoxide (141.8 g, 1.11 equiv.) was dissolved
in dry DMF (1 L). Diethyl malonate (266 mL, 1.54 equiv.) was added
over 5 minutes. Bromoacetaldehyde diethylacetal (172 mL, 1.14 mole)
was added over 5 minutes. The mixture was heated to 120.degree. C.
(internal temperature), and stirred at 120.degree. C. for 5 hours.
The mixture was allowed to cool to room temperature, poured into
water (5 L), and extracted with methyl tert-butyl ether (MTBE,
3.times.600 mL). The organic solution was dried over MgSO.sub.4,
filtered, concentrated, and distilled (0.5 mm, 95-140.degree. C.)
to yield the desired diester (244 g, 78%) as a colorless oil.
[0341] .sup.1H NMR (CDCl.sub.3) .delta. 1.19 (t, 6H), 1.28 (t, 6H),
2.22 (dd, 2H), 3.49 (m, 2H), 3.51 (t, 1H), 3.65 (m, 2H) 4.20 (qd,
4H), 4.54 (t, 1H).
b) Preparation of 4,4-diethoxy-2-(hydroxymethyl)-butanol
[0342] 16
[0343] LiBH.sub.4 (purchased solution, 2M in THF, 22.5 mL) and the
product of Example 14 step a) (5 g in 15 mL of THF, 18.1 mmol) were
combined and warmed to 60.degree. C. and stirred at 60.degree. C.
for 4 hours. The reaction mixture was allowed to cool to room
temperature and the reaction vessel was placed in a cool water
bath. Then triethanolamine (5.97 mL, 1 equiv.) was added at such a
rate that the temperature of the reaction mixture was maintained
between 20-25.degree. C. Brine (17.5 mL) was added at a rate such
that gas evolution was controlled and the mixture was stirred for
45 minutes at room temperature. The layers were separated, the
organic layer was washed with brine (2.times.15 mL). The combined
brine washes were extracted with MTBE (methyl tert-butyl ether,
3.times.20 mL). The combined organic extracts were evaporated and
the residue was dissolved in MTBE (50 mL) and washed with brine (25
mL). The brine layer was back-extracted with MTBE (3.times.25 mL).
The combined organic extracts were dried over Na.sub.2SO.sub.4,
filtered, and concentrated to yield the desired diol (3.36 g, 15.5
mmol, 97%) as a colorless oil.
[0344] .sup.1H NMR (CDCl.sub.3) .delta. 1.22 (t, 6H), 1.73 (dd,
2H), 1.92 (m, 1H), 2.67 (bs, 2H), 3.52 (m, 2H), 3.69 (m, 2H), 3.72
(m, 4H), 4.62 (t, 1H).
c) Preparation of (2R)-2-acetoxymethyl-4,4-diethoxy-butanol
[0345] 17
[0346] Into a 10 ml 1 neck round bottom flask was charged the
product of Example 14 step b) (3.84 g, 20 mmol), followed by
addition of vinyl acetate (2.6 g, 30 mmol) and finally Lipase PS 30
(69 mg, purchased from Amano, Lombard, Ill.). The mixture was
allowed to stir at ambient temperature for 16 hours. Progress of
the reaction was closely monitored by TLC (2/1 hexane-EtOAc;
stained with Ce.sub.2(SO.sub.4).sub.3 and charred on hot plate;
r.f. of diol is 0.1, monoacetate is 0.3, bis acetate is 0.75). The
reaction mixture was diluted with CH.sub.2Cl.sub.2 and filtered
through a 5 micron filter. The filter was washed with additional
CH.sub.2Cl.sub.2. The filtrate was then concentrated in vacuo to
afford the desired product.
d) Preparation of (2S)-2-acetoxymethyl-4,4-diethoxybutyl
toluenesulfonate
[0347] 18
[0348] Into a 100 mL 1-neck round bottom flask, equipped with a
magnetic stir bar and septum under N.sub.2 was charged the crude
product of Example 14 step c) (4.62 g, 19 mmol), dry
CH.sub.2Cl.sub.2 (20 mL) and Et.sub.3N (5.62 mL, 40 mmol). To this
solution was added tosyl chloride (4.76 g, 25 mmol). The resulting
mixture was stirred at ambient temperature for 4 hours. Charged
H.sub.2O (0.27 g, 15 mmol) and stirred vigorously for 4 hours. The
reaction mixture was diluted with 80 mL EtOAc and 50 mL H.sub.2O
and the aqueous layer was separated. To the organic layer was added
75 ml of a 5% aq. solution of KH.sub.2PO.sub.4. After mixing and
separation of the layers, the aqueous layer was removed. The
organic layer was washed with 50 mL of saturated NaHCO.sub.3
solution, dried over Na.sub.2SO.sub.4, filtered and concentrated in
vacuo to a constant weight of 7.40 g of the desired product.
[0349] .sup.1H NMR (CDCl.sub.3) .delta. 1.17 (t, 6H); 1.62 (m, 2H);
1.94 (s, 3H); 2.19 (m, 1H); 2.45 (s, 3H); 3.42 (m, 2H); 3.6 (m,
2H); 4.03 (m, 4H); 4.51 (t, 1H); 7.36 (d, 2H); 7.79 (d, 2H).
[0350] e) Preparation of 19
[0351] Into a 50 mL 1 neck round bottom flask was charged the
product of Example 14 step d) (3.88 g, 10 mmol), anhydrous DMF (20
mL), 2-amino-4-chloro-purine (2.125 g, 12.5 mmol) and
K.sub.2CO.sub.3 (4.83 g). The resulting suspension was stirred at
40.degree. C. under a N.sub.2 blanket for 20 hours. The mixture was
concentrated to remove most of the DMF on a rotary evaporator. The
residue was diluted with EtOAc (50 mL) and H.sub.2O (50 mL). The
reaction mixture was transferred to a separatory funnel, shaken and
the aqueous layer was separated. The aqueous layer was extracted
with EtOAc (25 mL). The organic layers were combined and washed
with 5% KH.sub.2PO.sub.4 (75 mL). The organic layer was separated
and washed with H.sub.2O (75 mL), brine (75 mL), dried over
Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford 3.95
g of crude product. The crude product was slurried with 40 mL of
methyl-t-butyl ether. This mixture was stirred overnight at
4.degree. C. and the mixture was filtered. The filtrate was
concentrated to afford 3.35 g of the product as an oil (containing
2.6 g of the desired product based upon HPLC analysis).
[0352] 300 MHz .sup.1H NMR (CDCl.sub.3) .delta. 1.19 (m, 6H); 1.69
(2H); 1.79 (s, 1H); 2.03 (s, 3H); 2.52 (m, 1H); 3.48 (m, 2H); 3.62
(m, 2H); 4.04 (m, 2H); 4.16 (m, 2H); 4.61 (t, 1H); 5.12 (bs, 2H);
7.81 (s, 1H).
[0353] f) Preparation of 20
[0354] Into a 500 mL 1 neck round bottom flask was charged benzyl
alcohol (136 mL), cooled to 0.degree. C., followed by portionwise
addition of KO-t-Bu (36 g, 321 mmol). The temperature was allowed
to warm to 40.degree. C., and the mixture was stirred 20 minutes.
To this mixture was added at 0.degree. C. the crude product of
Example 14 step e) (24.7 g, 64.2 mmol) dissolved in 25 mL anhydrous
THF and benzyl alcohol (30 mL). The temperature was allowed to
slowly warm to 8.degree. C. over 2 hours. The reaction mixture was
poured into 500 mL ice and was extracted with 500 mL MTBE. The
organic layer was washed with 250 mL of brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford 193
g of a benzyl alcohol solution of the desired product. HPLC
analysis indicated that the solution contained 25.96 g of the
desired product.
[0355] MHz .sup.1H NMR (CDCl.sub.3) .delta. 1.22 (m, 6H); 1.55
(2H); 2.18 (m, 1H); 3.15 (m, 1H); 3.40 (m, 1H); 3.51 (m, 2H); 3.70
(m, 2H); 4.25 (m, 2H); 4.63 (t, 1H); 4.90 (bs, 2H); 5.25 (m, 1H);
5.58 (s, 2H); 7.35 (m, 3H); 7.51 (m, 2H); 7.72 (s, 1H).
MS=(M+H).sup.+=416 (Cl).
[0356] g) Preparation of 21
[0357] Into a 100 mL 1 neck round bottom flask was charged the
crude product of Example 14 step f) (9.65 g of the benzyl alcohol
solution, containing 1.30 g, 3.13 mmol of the product of Example
14, step f) dissolved in absolute EtOH (20 mL). To this was added
0.45 g of 10% Pd/C slurried in 5 mL absolute EtOH. The reaction
flask was evacuated and charged with H.sub.2 three times with a
balloon of H.sub.2. The reaction flask was pressurized with 1 atm.
H.sub.2 and the reaction mixture was stirred overnight. The
reaction mixture was filtered through a pad of diatomaceous earth
to remove Pd/C. The volatiles were removed in vacuo. The residue
was mixed with 25 mL of isopropyl acetate and then concentrated in
vacuo. The residue was diluted with EtOAc (10 mL), seeded with the
desired product, heated to reflux and then CH.sub.3CN (2 mL) and
MTBE (35 ml) were added. The mixture was stirred for 30 minutes.
The precipitate was filtered and dried to a constant weight of 600
mg of the desired product.
[0358] MHz .sup.1H NMR (d.sub.6-DMSO) .delta. 1.16 (m, 6H); 1.45
(m, 1H); 1.61 (m, 1H); 2.16 (m, 1H); 3.45 (m, 2H); 3.40 (m, 1H);
3.62 (m, 2H); 4.02 (m, 2H); 4.53 (t, 1H); 4.85 (t, 1H); 6.55 (bs,
1H); 7.75 (s, 1H). MS=(M+H).sup.+=416 (Cl).
[0359] h) Preparation of 22
[0360] Into a 25 mL 1 neck round bottom flask was charged the
product of Example 14 step g) (0.650 g, 2.0 mmol), pyridine (4 mL)
and CH.sub.2Cl.sub.2 (2 mL), DMAP (10 mg). The mixture was cooled
to -5.degree. C. and stearoyl chloride (790 mg, 2.6 mmol) dissolved
in CH.sub.2Cl.sub.2 (0.5 mL) was added over 5 minutes. The
resulting mixture was stirred 16 hours at -5.degree. C. Absolute
EtOH (0.138 g, 3.0 mmol) was added and the mixture was stirred an
additional 1 hour. The reaction mixture was concentrated in vacuo.
Toluene (30 mL) was added to the residue and then the mixture was
concentrated in vacuo. Again, toluene (30 mL) was added to the
residue and then the mixture was concentrated in vacuo. To the
residue was added 1% KH.sub.2PO.sub.4 (25 mL) and this mixture was
extracted with CH.sub.2Cl.sub.2 (60 mL). The organic layer was
separated and was dried over Na.sub.2SO.sub.4, filtered and
concentrated in vacuo to a constant weight of 1.65 g. The crude
product was chromatographed on 40 g of SiO.sub.2, eluting with 95/5
CH.sub.2Cl.sub.2-EtOH, affording 367 mg of the desired product.
[0361] MHz .sup.1H NMR (CDCl.sub.3) .delta. 0.89 (t, 3H); 1.26 (m,
30H); 1.65 (m, 3H); 2.32 (m, 1H); 3.45 (m, 1H); 3.60 (m, 2H); 4.08
(m, 2H); 4.60 (m, 1H); 6.0 (bs, 2H); 7.53 (s, 1H).
[0362] i) Preparation of 23
[0363] Into a 25 mL 1 neck round bottom flask was charged the
product of Example 14, step h) (0.234 g, 0.394 mmol) dissolved in
THF (1.7 mL). To this solution was added triflic acid (0.108 g) in
H.sub.2O 180 mg. The mixture was stirred overnight at room
temperature. To the reaction mixture was added saturated
NaHCO.sub.3 solution (10 mL), THF (5 mL), CH.sub.2Cl.sub.2 (2 mL)
and NaBH.sub.4 (0.10 g). This mixture was stirred for 30 minutes.
To the reaction mixture was added a 5% solution of KH.sub.2PO.sub.4
(30 mL). This mixture was extracted with 2.times.15 ml of
CH.sub.2Cl.sub.2. The organic layers were combined and dried over
Na.sub.2SO.sub.4, filtered and concentrated in vacuo to a constant
weight of 207 mg. This material was recrystallized from EtOAc (8
mL) and CH.sub.3CN (0.5 mL) affording 173 mg of the desired
product.
[0364] 300 MHz .sup.1H NMR (d.sub.6-DMSO) .delta. 0.82 (t, 3H);
1.19 (m, 30H); 1.41 (m, 4H); 2.19 (t, 2H); 2.32 (m, 1H); 3.40 (m,
2H); 3.9 (m, 4H); 4.49 (m, 1H); 6.4 (bs, 2H); 7.61 (m, 1.5H); 9.55
(m, 0.5H).
EXAMPLE 15
Alternative preparation of
(R)-9-[4-(N-tert-butyloxycarbonyl-L-valyloxy)-2-
-(stearoyloxymethyl)butyl]guanine
[0365]
(R)-9-[2-(Stearoyloxymethyl)-4-(t-butyldiphenylsilyloxy)butyl]guani-
ne (45 g) and THF (950 ml) were combined in a 2 L flask. Then
Boc-L-valine (3.22 g, 0.25 eq) was added, followed by
tetrabutylammonium fluoride (1 M in THF, 89.05 mL) over 10 minutes.
The clear reaction mixture was stirred at room temperature for 2
hours and 50 minutes with monitoring of the reaction progress by
TLC (90/10 CH.sub.2Cl.sub.2/MeOH).
[0366] To the reaction mixture was added Boc-L-valine (35.43 g,
2.75 eq), DCC (36.67 g, 2.75 eq) and dimethylaminopyridine (1.1 g,
0.15 eq) in THF (25 ml). The reaction mixture was stirred at room
temperature for 24 hours. DCU was filtered off and washed with
CH.sub.2Cl.sub.2. The filtrate was concentrated, and the residue
was taken up in 2 litres of CH.sub.2Cl.sub.2 and washed with 2 L of
1/2 saturated sodium bicarbonate and brine solutions. On drying and
evaporation, approximately 100 g of crude product was obtained. The
material was purified by silica chromatography (6000 ml of silica)
using 3% MeOH/CH.sub.2Cl.sub.2 to 5% MeOH/CH.sub.2Cl.sub.2 to
obtain 38.22 mg of the desired product.
EXAMPLE 16
Alternative preparation of
(R)-9-[2-(stearoyloxymethyl)-4-(L-valyloxy) butyl]guanine
a)
(R)-9-[2-Hydroxymethyl)-4-(t-butyldiphenylsilyloxymethyl)butyl]guanine
[0367] H2G (450.0 g, 1.78 mol) and N,N dimethylformamide (6.4 kg)
were charged into a Bucchi evaporator and the mixture warmed to
dissolve the solid. The solution was concentrated to dryness under
vauum at no more than 90.degree. C. The resulting powder was
transferred to a 22 litre flask with stirrer, addition funnel and
and temperature probe. N,N-dimethylformamide (1.7 kg) was added
followed by pyridine (3.53 kg). The resulting suspension was cooled
to -10.degree. C. under nitrogen and stirred at
-5.degree..+-.5.degree. C. as t-butylchlorodiphenylsilane (684 g,
2.49 mol) was added dropwise. The resulting mixture was stirred at
-5.degree..+-.5.degree. C. until the reaction was complete (as
monitored by TLC (10:1 methylene chloride/methanol) and HPLC
(4.6.times.250 mm Zorbax RxC8 (5 micron); 60:40 acetonitrile-aq.
NH.sub.4OAC (0.05 M) at 1.5 ml/min; UV detection at 254 nm)). Water
(16 kg) was added and the mixture was stirred for 30 minutes to
precipitate the product, then the mixture was cooled to 0.degree.
C. for 30 minutes. The solid was isolated by filtration and the
product cake was washed with cold water and sucked dry with air to
provide the crude product as an off-white solid. The crude solid
was taken up in pydridine (3 kg) and concentrated under vacuum at
60.degree. C. to remove water. The dry solid residue was slurried
with methanol (10 kg) at 60.degree. C. for 1-2 hours and filtered
while hot. The filtrate was concentrated under vacuum and the solid
residue was refluxed with isopropyl acetate (7 kg) for 30 minutes.
The mixture was cooled to 20.degree. C. and filtered. The filter
cake was dried under vacuum at 50.degree. C. to provide the title
compound as a white solid (555 g).
b)
(R)-9-[2-(Stearoyloxymethyl)-4-(t-butyldiphenylsilyloxy)butyl]guanine
[0368] The product of Example 16, step a) (555 g, 1.113 mol) was
charged to a 50 litre Buchi evaporator. Pyridine (2.7 kg) was added
dropwise to dissolve the solid and the mixture was distilled to
dryness under vacuum at 60.degree. C. The residue was taken up in
fresh pyridine (2.7 kg) and transferred to a 22 litre flask with
stirrer, addition funnel and temperature probe. The solution was
cooled to -5.degree. C. under nitrogen. A solution of stearoyl
chloride (440 g, 1.45 mol) in methylene chloride (1.5 kg) was added
so as to maintain a temperature below 0.degree. C.
4-(N,N-dimethylamino)pyridine (15 g, 0.12 mol) was added and the
mixture was stirred at -5-0.degree. C. for 2-4 hours until
conversion was complete (as monitored by TLC (10:1 methylene
chloride/methanol) and HPLC (4.6.times.250 mm Zorbax RxC8 (5
micron); 60:40 acetonitrile-aq. NH.sub.4OAc (0.05 M) at 1.5 ml/min;
UV detection at 254 nm)). At the end of the reaction, acetonitrile
(8.7 kg) was added and the mixture was stirred for not less than 15
minutes to precipitate the product. The slurry was cooled to
0.degree. C. for 2 hours and the solid isolated by filtration and
the filter cake washed with acetonitrile (2 kg). The desired
product was obtained as a white solid (775 g).
c) (R)-9-[4-Hydroxy-2-(stearoyloxymethyl)butyl]guanine
[0369] A solution of the product of Example 16, step b) (765 g,
0.29 mol) in tetrahydrofuran (10 kg) was prepared in a reactor. A
solution of tetra(n-butyl)ammonium fluoride in tetrahydrofuran (1.7
kg of 1 M solution, 1.7 mol) was added and the resulting clear
solution was stirred at 200.+-.5.degree. C. for 4 hours. Water (32
kg) was added and the resulting slurry was stirred for 1 hour and
then cooled to 0.degree. C. for 30 minutes. The precipitate was
isolated by filtration and the filter cake was washed successively
with water (10 kg) and acetonitrile (5 kg). After drying under
vacuum at 25.degree. C., 702 g of crude product was obtained. The
crude product was dissolved in refluxing THF (4.2 kg) and water
(160 g), then cooled to 40.degree. C. and treated with methylene
chloride (14.5 kg). The mixture was allowed to cool to
25.+-.5.degree. C. for 1 hour, then it was cooled to
5.degree..+-.5.degree. C. for 1 hour to complete precipitation. The
slightly off-white powder was isolated by filtration and dried
under vacuum at 40.degree. C. to yield the desired product (416
g).
d)
(R)-9-[4-(N-Cbz-L-valyloxy)-2-(stearoyloxymethyl)butyl]guanine
[0370] A solution of N-Cbz-L-valine (169 g, 0.67 mol) in dry THF
(750 ml) was prepared in a 2 litre flask with mechanical stirrer,
thermometer and addition funnel. A solution of
dicyclohexylcarbodiimide (69.3 g, 0.34 mol) in THF (250 ml) was
added over 5 minutes and the resulting slurry was stirred at
20.degree.+5.degree. C. for 2 hours. The slurry was filtered and
the filter cake was washed with THF (300 ml). The filtrate and wash
were charged to a 3 litre flask with stirrer and thermometer. The
product of Example 16, step c) (116 g, 0.22 mol) was added as a
solid, with a rinse of THF (250 ml). 4-(N,N-dimethylamino)pyridine
(2.73 g, 0.022 mol) was added and the white slurry stirred at
20.degree..+-.5.degree. C. Within 15 minutes, the solids were all
dissolved and the reaction was complete within 1 hour (as
determined by HPLC: 4.6.times.250 mm Zorbax RxC8 column; 85:15
acetonitrile-0.2% aq. HClO.sub.4 at 1 ml/min.; UV detection at 254
nm; starting material elutes at 4.1 min. and product elutes at 5.9
min.). The reaction was quenched by addition of water (5 ml) and
the solution was concentrated under vacuum to leave a light yellow
semisolid. This was taken up in methanol (1.5 litres) and warmed to
reflux for 30 minutes. The solution was cooled to 25.degree. C. and
the precipitate was removed by filtration. The filtrate was
concentrated under vacuum to leave a viscous, pale yellow oil.
Acetonitrile, (1 L) was added and the resulting white suspension
was stirred at 20.degree..+-.5.degree. C. for 90 minutes. The crude
solid product was isolated by filtration, washed with acetonitrile
(2.times.100 ml) and air-dried overnight to provide the desired
product as a waxy, sticky solid (122 g). This was further purified
by crystallization from ethyl acetate (500 ml) and drying under
vacuum at 30.degree. C. to provide the desired product as a white,
waxy solid (104 g).
e) (R)-9-[4-(L-valyloxy)-2-(stearoyloxymethyl)butyl]guanine
[0371] A solution of the product of Example 16, step d), (77 g) in
warm (40.degree. C.) ethanol (2.3 L) was charged to an
hydrogenation reactor with 5% Pd--C (15.4 g). The mixture was
agitated at 40.degree. C. under 40 psi hydrogen for 4 hours,
evacuated and hydrogenated for an additional 4-10 hours. The
catalyst was removed by filtration and the filtrate was
concentrated under vacuum to provide a white solid. This was
stirred with ethanol (385 ml) at 25.degree. C. for 1 hour, then
cooled to 0.degree. C. and filtered. The filter cake was dried with
air, then under vacuum at 35.degree. C. to yield the title compound
as a white powder (46 g).
EXAMPLE 17
(R)-9-[2-(L-Valyloxymethyl)-4-(stearoyloxy)butyl]guanine
a) (R)-9-[2-Hydroxymethyl-4-(stearoyloxy)butyl]guanine
[0372] H2G (506 mg; 2.0 mmol) was dissolved in dry
N,N-dimethylformamide (40 ml) with pyridine (400 mg; 5.06 mmol) and
4-dimethylaminopyridine (60 mg; 0.49 mmol). Stearoyl chloride (1500
mg; 4.95 mmol) was added and the mixture kept overnight at room
temperature. Most of the solvent was evaporated in vacuo, the
residue stirred with 70 ml ethyl acetate and 70 ml water, and the
solid filtered off, washed with ethyl acetate and water and dried
to yield 680 mg of crude product. Column chromatography on silica
gel (chloroform:methanol 15:1) gave pure title compound as a white
solid.
[0373] .sup.1H NMR (DMSO-d.sub.6) .delta. 0.86 (t, 3H); 1.25 (s,
28H); 1.51 (qui, 2H); 1.62 (m, 2H); 2.06 (m, 1H); 2.23 (t, 2H);
3.34 (d, 2H); 3.96 (ABX, 2H); 4.07 (dd, 2H); 6.30 (br s, 2H); 7.62
(s, 1H); 10.45 (s, 1H).
[0374] .sup.13C NMR (DMSO-d.sub.6) .delta. 13,8 (C18); 22.0 (C17);
24.4 (C3); 27.7 (C3'); 28.4-28.8 (C.sub.4-6, C15); 28.9
(C.sub.7-14); 31.2 (C16); 33.5 (C2); 38.0 (C2'); 44.0 (C1');
60.6/61.8 (C4', C2"); 116.5 (guaC5); 137.7 (guaC7); 151.4 (guaC4);
153.5 (guaC2); 156.7 (guaC6); 172.7 (COO).
b)
(R)-9-[2-(N-Boc-L-valyloxymethyl)-4-(stearoyloxy)butyl]guanine
[0375] A mixture of N-Boc-L-valine (528 mg; 2.1 mmol) and
N,N'-dicyclohexyl carbodiimide (250 mg; 1.21 mmol) in
dichloromethane (20 ml) was stirred over night at room temperature,
dicyclohexylurea filtered off and extracted with a small volume of
dichloromethane, and the filtrate evaporated in vacuo to a small
volume. (R)-9-[2-Hydroxymethyl-4-- (stearoyloxy)butyl]guanine (340
mg; 0.654 mmol), 4-dimethylaminopyridine (25 mg; 0.205 mmol), and
dry N,N-dimethylformamide (15 ml) were added and the mixture was
stirred for 4 h at 50.degree. C. under N.sub.2. The solvent was
evaporated in vacuo to a small volume. Column chromatography on
silica gel, then on aluminum oxide (ethyl acetate:methanol:water
15:2:1 as eluent) gave 185 mg (39%) pure title compound as a white
solid.
[0376] .sup.1H NMR (CHCl.sub.3) .delta. 0.85-1.0 (m, 9H)
18-CH.sub.3, CH(CH.sub.3).sub.2; 1.25 (s, 28H) 4-17-CH.sub.2; 1.44
(s, 9H) t-Bu; 1.60 (qui, 2H) 3-CH.sub.2; 1.74 (qua, 2H)
3'-CH.sub.2; 2.14 (m, 1H) 2'-CH; 2.29 (t, 2H) 2-CH.sub.2; 2.41 (m,
1H)CH(CH.sub.3).sub.2; 4.1-4.3 (m, 6H)C1'-CH.sub.2, C2"-CH.sub.2,
C4-CH.sub.2; 5.4 (d, 1H) .alpha.CH; 6.6 (br s, 2H) guaNH.sub.2;
7.73 (s, 1H) guaH8; 12.4 (br s).
[0377] .sup.13C NMR (CHCl.sub.3) .delta. 13,9 (C18); 17,5/18.9 (2
Val CH.sub.3); 22.4 (C17); 24.7 (C3); 28.1 (C3'); 28.9-29.3
(C.sub.4-6, C15); 29.4 (C.sub.7-14); 30.7 (Val .alpha.C); 31.7
(C16); 34.0 (C2); 35.9 (C2'); 43.9 (C1'); 58.7 (Val .alpha.C);
61.4/63.6 (C4', C2"); 79.9 (CMe.sub.3); 116.4 (guaC5); 137.9
(guaC7); 151.7 (guaC4); 153.7 (guaC2); 155.7 (CONH); 158.8 (guaC6);
172.1 (CHCOO); 173.5 (CH.sub.2COO).
c) (R)-9-[2-(L-Valyloxymethyl)-4-(stearoyloxy)butyl]guanine
[0378] Chilled trifluoroacetic acid (2.0 g) was added to
(R)-9-[2-(N-Boc-L-valyloxymethyl).sub.4-(stearoyloxy)butyl]guanine
(180 mg; 0.25 mmol) and the solution kept at room temperature for 1
h, evaporated to a small volume, and lyophilized repeatedly with
dioxane until a white amorphous powder was obtained. The yield of
title compound, obtained as the trifluoracetate salt, was
quantitative.
[0379] .sup.1H NMR (DMSO-d.sub.6) .delta. 0.87 (t, 3H) 18-CH.sub.3,
0.98 (dd, 6H)CH(CH.sub.3).sub.2; 1.25 (s, 28H) 4-17-CH2; 1.50 (qui,
2H) 3-CH.sub.2; 1.68 (qua, 2H) 3'-CH.sub.2; 2.19 (m, 1H) 2'-CH;
2.26 (t, 2H) 2-CH.sub.2; 2.40 (m, 1H)CH(CH.sub.3).sub.2; 3.94.25
(m, 7H)C1'-CH.sub.2, C2"-CH.sub.2, C4-CH.sub.2, .alpha.CH; 6.5 (br
s, 2H) guaNH.sub.2; 7.79 (s, 1H) guaH8; 8.37 (br s, 3H)NH.sub.3+;
10.73 (br s, 1H) guaNH.
[0380] .sup.13C NMR (DMSO-d.sub.6) .delta. 14.2 (C18); 17.9/18.3 (2
Val CH3); 22.3 (C17); 24.6 (C3); 27.7 (C3'); 28.7-29.1 (C.sub.4-6,
C15); 29.2 (C.sub.7-14); 29.5 (Val .beta.C); 31.5 (C16); 33.7 (C2);
35.0 (C2'); 44.1 (C1'); 57.6 (Val .alpha.C); 61.6/65.2 (C4', C2");
116.1 (guaC5); 116.3 (qua, J=290 Hz, CF3); 137.9 (guaC7); 151.5
(guaC4); 154.0 (guaC2); 156.7 (guaC6); 158.3 (qua, J=15 Hz,
CF.sub.3COO) 169.1 (CHCOO); 173.1 (CH.sub.2COO).
EXAMPLE 18
Alternative preparation of
(R)-9-[2-hydroxymethyl-4-(stearoyloxy)butyl]gua- nine
[0381] H2G (7.60 g, 30 mmol) was heated to solution in dry DMF (200
ml). The solution was filtered to remove solid impurities, cooled
to 20.degree. C. (H2G cystallized) and stirred at that temperature
during addition of pyridine (9.0 g, 114 mmol),
4-dimethylaminopyridine (0.46 g, 3.75 mmol) and then, slowly,
stearoyl chloride (20.0 g, 66 mmol). Stirring was continued at room
temperature overnight. Most of the solvent was then evaporated off
in vacuo, the residue stirred with 200 ml ethyl acetate and 200 ml
water and the solid filtered off, washed with ethyl acetate and
water and dried to yield crude product. As an alternative to
recrystallization, the crude product was briefly heated to almost
boiling with 100 ml of ethyl acetate:methanol:water (15:2:1) and
the suspension slowly cooled to 30.degree. C. and filtered to leave
most of the 2" isomer in solution (the 2" isomer would crystallize
at lower temperature). The extraction procedure was repeated once
more to yield, after drying in vacuo, 6.57 g (42%) of almost isomer
free product.
EXAMPLE 19
Preparation of crystalline
(R)-9-[2-stearoyloxymethyl)-4-(L-valyloxy)butyl- ]guanine
[0382] The product of Example 16, step c) (20.07 g, 32.5 mmol) was
dissolved in absolute ethanol (400 ml) with heating, filtered, and
further diluted with ethanol (117.5 ml). To this solution was added
water (HPLC grade, 103.5 ml), and the mixture was allowed to cool
to 3540.degree. C. After the mixture was cooled, water (HPLC grade,
931.5 ml) was added at a constant rate over 16 hours with efficient
stirring. After all the water was added, stirring was continued for
4 hours at room temperature. The resulting precipitate was filtered
through paper and dried under vacuum at room temperature to obtain
the title compound as a white, free flowing crystalline powder
(19.43 g, 97%), m pt 169-170.degree. C.
EXAMPLE 20
9-R-(4-Hydroxy-2-(L-valyloxymethyl)butyl) guanine
[0383] a) To a solution of
9-R-(4-(tert-butyldiphenylsilyloxy)-2-(hydroxym-
ethyl)butyl)guanine (695 mg, 1.5 mmole) in DMF (30 ml) were added
N-Boc-L-Valine (488 mg, 2.25 mmole), 4-dimethylamino pyridine (30
mg, 0.25 mmole) and DCC (556 mg, 2.7 mmole). After 16 hr, the
reaction was recharged with N-Boc-L-valine (244 mg) and DCC (278
mg), and was kept for an additional 5 hours. The reaction mixture
was filtered through Celite and poured into sodium hydrogen
carbonate aqueous solution, and then it was extracted with
dichloromethane. The organic phase was evaporated and purified by
silica gel column chromatography, giving 950 mg of the N-protected
monoamino acyl intermediate.
[0384] b) The above intermediate (520 mg, 0.78 mmole) was dissolved
in THF (15 ml). To the solution was added hydrogen fluoride in
pyridine (70%/30%, 0.34 ml). After two days, the solution was
evaporated and coevaporated with toluene. Purification by silica
gel column chromatography gave 311 mg of the protected monoamino
acyl compound.
[0385] .sup.1H-NMR (DMSO-d.sub.6): .delta. 10.41 (s, 1H), 7.59
(1H), 6.26 (br s, 2H), 4.32 (t, 1H), 3.95 (m, 5H), 3.46 (m, 2H),
2.41 (m, 1H), 2.06 (m, 1H), 1.45 (m, 2H), 1.39 (s, 9H), 0.90 (d,
6H).
[0386] c) The product of step b) (95 mg, 0.21 mmole) was treated
with a mixture of trifluoroacetic acid (4 ml) and dichloromethane
(6 ml) for 1 hr. The solution was evaporated and freeze-dried, to
give 125 mg of the unprotected monoaminoacyl product.
[0387] .sup.1H-NMR (D.sub.2O): .delta. 8.88 (s, 1H), 4.32 (m, 4H),
3.96 (d, 1H), 3.68 (m, 2H), 2.63 (m, 1H), 2.22 (m, 1H), 1,73 (m,
2H), 1.00 (m, 6H).
EXAMPLE 21
(R)-9-(2-Hydroxymethyl-4-(L-isoleucyloxy)butyl) guanine
[0388] a) To a solution of
(R)-9-(2-hydroxymethyl-4-hydroxybutyl)guanine (2.53 g, 10 mmole) in
DMF (250 ml) were added N-Boc-L-isoleucine (2.77 g, 12 mmole),
4-dimethylaminopyridine (61 mg, 0.6 mmole) and DCC (3.7 g, 18
mmole). After reaction for 16 hr at 0.degree.(C, N-Boc-L-isoleucine
(1.3 g) and DCC (1.8 g) were recharged, and the reaction was kept
overnight at room temperature. The reaction mixture was filtered
through Celite and the filtrate was evaporated and purified by
silica gel column chromatography, giving 1.25 g of the N-protected
monoamino acyl intermediate.
[0389] .sup.1H-NMR (DMSO-d.sub.6): 610.56 (s, 1H), 7.62 (s, 1H),
6.43 (s, 2H), 4.75 (t, 1H), 4.15-3.80 (m, 5H), 3.25 (m, 2H) 2.05
(m, 1H), 1.80-1-05 (m, 14H), 0.88 (m, 6H).
[0390] b) The intermediate from step a) (100 mg, 0.21 mmole) was
treated with trifluoroacetic acid (3 m) and for 30 min at 0.degree.
C. The solution was evaporated and freeaze-dried, to give the
titled unprotected mono-aminoacyl product in quantitative
yield.
[0391] .sup.1H-NMR (DMSO-d.sub.6+D.sub.2O): .delta. 8.72 (s, 1H),
4.15 (m, 4H), 3.90 (d, 1H), 3.42 (m, 2H), 2.09 (m, 1H), 1.83 (m,
1H), 1.61 (m, 2H), 1.15 (m, H), 0.77 (d, 3H), 0.71 (t, 3H).
EXAMPLE 22
(R)-9-[2-Hydroxymethyl-4-(L-valyloxy)butyl]guanine
[0392] The product of Example 1, step a) was deprotected with
trifluoroaacetic acid in the same manner as Example 1, step c)
[0393] .sup.1H-NMR (250 MHz, DMSO-d.sub.6): 61.04 (dd, 6H),
1.55-1.88 (m, 2H), 2.21 (m, 2H), 3.48 (m, 2H), 4.00 (m, 1H), 4.13
(m, 2H), 4.34 (t, 2H), 6.9 (br s, 2H), 8.21 (s, 1H), 8.5 (br s,
3H), 11.1 (br s, 1H).
EXAMPLE 23
(R)-9-[2-(L-Valyloxymethyl)-4-(valyloxy)butyl]guanine
a)
(R)-9-[4-(N-Boc-L-valyloxy)-2-(N-Boc-L-valyloxymethyl)butyl]guanine
[0394] Application of the technique described in Example 1, step
a), but using 2.7 eqs, 0.28 eqs, and 3.2 eqs of N-Boc-L-valine,
DMAP, and DCC, respectively, resulted in the title compound.
[0395] .sup.1H NMR (250 MHz, CDCl.sub.3) .delta.0.95 (m, 12H), 1.42
(br s, 18H), 1.8 (m, 2H), 2.14 (m, 2H), 2.47 (m, 1H), 4.0-4.4 (m,
8H), 6.5 (br s, 2H), 7.67 (s, 1H).
b) (R)-9-[4-(L-Valyloxy)-2-(L-valyloxymethyl)butyl]guanine
[0396] The titled compound was obtained as the
tris-trifluoroacetate salt from the intermediate of Example 23 step
a) by deprotection in a manner analogous to Example 1 step c).
[0397] .sup.1H NMR (250 MHz, D.sub.2O) .delta. 1.0 (m, 12H), 1.89
(m, 2H), 2.29 (m, 2H), 2.62 (m, 1H), 4.02 (dd, 2H), 4.38 (m, 6H),
4.89 (br s, ca. 10H), 8.98 (s, 1H).
EXAMPLE 24
(R)-9-[4-hydroxy-2-(stearoyloxymethyl)butyl]guanine
[0398] The titled compound is prepared according to steps a) to c)
of Example 7.
[0399] .sup.1H NMR (250 MHz, DMSO-d.sub.6): .delta. 10.52 (s, 1H),
7.62 (s, 1H), 6.39 (s, 2H), 4.50 (t, 1H), 3.93 (m, 4H), 3.42 (m,
2H), 2.45 (m, 1H), 2.23 (t, 2H), 1.48 (m, 4H), 1.22 (s, 28H), 0.89
(t, 3H)
EXAMPLE 25
(R)-9-[2-Hydroxymethyl-4-stearoyloxy)butyl]guanine
[0400] The titled compound is prepared by the procedure of Example
17, step a).
[0401] .sup.1H NMR (DMSO-d.sub.6) .delta. 0.86 (t, 3H); 1.25 (s,
28H); 1.51 (qui, 2H); 1.62 (m, 2H); 2.06 (m, 1H); 2.23 (t, 2H);
3.34 (d, 2H); 3.96 (ABX, 2H); 4.07 (dd, 2H); 6.30 (br s, 2H); 7.62
(s, 1H); 10.45 (s, 1H).
EXAMPLE 26
Alternative preparation of
(R)-9-[2-stearoyloxymethyl)-4-(L-valyloxy)butyl- ]guanine
a)
(R)-9-[4-N-benzyloxycarbonyl-L-valyloxy)-2-(hydroxymethyl)-butyl]guanin-
e
[0402] Dry H2G (252 mg, 1 mmol), 4-dimethylaminopyridine (122 mg, 1
mmol) and N-Cbz-L-valine p-nitrophenyl ester (408 mg, 1.1 mmol)
were dissolved in dry dimethyl formamide (16 ml). After stirring at
23.degree. C. for 30 hours, the organic solvent was removed and the
residue carefully chromatographed (silica, 2%-7% methanol/methylene
chloride) to afford the desired product as a white solid (151 mg,
31%).
b)
(R)-9-[(4-N-benzyloxycarbonyl-L-valyloxy)-2-(stearoyloxymethyl)-butyl]g-
uanine
[0403] A solution of stearoyl chloride (394 mg, 1.3 mmol) in dry
methylene chloride (2 ml) was added slowly dropwise under nitrogen
to a solution of the product of step a) (243 mg, 1 mmol) and
4-dimethylaminopyridine (20 mg) in dry pyridine (5 ml) at
-5.degree. C. The reaction mixture was stirred at that temperature
for 12 hours. Methanol (5 ml) was added and the reaction stirred
for 1 hour. After removal of the solvent, the residue was
triturated with acetonitrile and chromatographed (silica, 0-5%
methanol/methylene chloride) to afford the desired product (542 mg,
72%).
c) (R)-9-[2-stearoyloxymethyl)-4-(L-valyloxy)butyl]guanine
[0404] The product of step b) (490 mg, 1 mmol) was dissolved in
methanol (30 ml) and 5% Pd/C (100 mg) added. A balloon filled with
hydrogen was placed on top of the reaction vessel. After 6 hours at
23.degree. C., TLC showed the absence of starting material. The
reaction mixture was filtered through a 0.45 micron nylon membrane
to remove the catalyst and the solvent was removed to afford the
desired product as a white solid (350 mg, 99%) which was identical
(spectral and analytical data) to Example 16.
EXAMPLE 27
Alternative preparation of
(R)-9-(4-hydroxy-2-(L-valyloxymethyl)butyl)guan- ine
[0405] (R)-9-(4-(L-valyloxy)-2-(L-valyloxymethyl)butyl)guanine from
Example 23 step b) (100 mg, 0,126 mmole) was dissolved in 0.1 N
NaOH aqueous solution (6.3 ml, 0.63 mmole) at room temperature. At
intervals, an aliquot was taken and neutralized with 0.5 N
trifluoroacetic acid. The aliquots were evaporated and analyzed by
HPLC to monitor the progress of the reaction. After 4 hours, 0.5 N
trifluoroacetic acid solution (1.26 ml, 0.63 mmole) was added to
the solution and the reaction mixture was evaporated. The desired
product was purified by HPLC, (YMC, 50.times.4.6 mm, gradient 0.1%
TFA+0-50% 0.1% TFA in acetonitrile, in 20 minutes, UV detection at
254 nm. Yield: 13.6%
[0406] .sup.1H-NMR (D.sub.2O): .delta. 8.81 (s, 1H), 4.36 (m, 4H),
4.01 (d, 1H), 3.74 (m, 2H), 2.64 (m, 1H), 2.25 (m, 1H), 1.73 (m,
2H), 1.03 (dd, 6H).
EXAMPLE 28
Alternative preparation of
(R)-9-(2-hydroxymethyl-4-(L-valyloxy)butyl)guan- ine
[0407] HPLC separation of the reaction solution from Example 27
gave the titled compound in 29.2% yield.
[0408] .sup.1H-NMR (DMSO-d.sub.6): .delta. 8.38 (s, 3H), 8.26 (s,
1H), 6.83 (br s, 2H), 4.23 (m, 2H), 4.06 (m, 2H), 3.91 (m, 1H),
3.40 (m, 2H), 2.19 (m, 2H), 1.8-1.40 (m, 2H), 0.95 (dd, 6H).
EXAMPLE 29
(R)-9-[(2-stearoyloxymethyl)-4-(L-valyloxy)butyl]guanine
monohydrochloride
[0409] The product of Example 16, step d) (360 mg, 0.479 mmol) was
dissolved in a mixture of methanol (10 ml) and ethyl acetate (10
ml). To the solution was added 10% Pd/C (100 mg) and 1 N HCl (520
microlitres). The reaction mixture was stirred at room temperature
for 2 hours under 1 atm. H.sub.2. The reaction mixture was filtered
and the solvent evaporated from the filtrate to provide the desired
product as a crystalline solid (300 mg).
EXAMPLE 30
Alternative preparation of
(R)-9-[(2-stearoyloxymethyl)-4-(L-valyloxy)buty- l]guanine
a) Preparation of
(R)-2-Amino-6-chloro-9-[4,4-diethoxy-2-(hydroxymethyl)bu-
tyl]purine
[0410] The product of Example 14, step e) (200 g) was dissolved in
methanol (670 mL) and 20% aqueous K.sub.2CO.sub.3 (43 g
K.sub.2CO.sub.3 in 166 mL H.sub.2O) was added. The mixture was
stirred at 25.+-.5.degree. C. for 30 minutes. The reaction mixture
was then cooled to 0-5.degree. C. for about 20 minutes, when a
precipitate formed. Water (500 mL) was added and the slurry was
mixed at 5.+-.5.degree. C. for 15 minutes. The resulting solid was
isolated by filtration and the filter cake was washed with water
(100 mL) and dried under vacuum at 20.degree. C. to provide the
desired product as a pale yellow powder (81 g). m.p.
156-158.degree. C.
[0411] 300 MHz .sup.1H NMR (DMSO-d.sub.6) .delta. 1.04 (m, 6H);
1.36 (m, 1H); 1.55 (m, 1H); 2.10 (m, 1H); 3.40 (m, 6H); 4.06 (m,
2H); 4.48 (t, 1H); 4.78 (t, 1H); 6.93, (br s, 2H); 8.10 (s,
1H).
b) Preparation of
(R)-9-[4,4-diethoxy-2-(hydroxymethyl)butyl]guanine
[0412] To the product of Example 30, step a) (22.5 kg, 65.4 moles)
was added an aqueous solution of KOH (prepared by dissolving 12.9
kg of KOH in 225 kg of water). This mixture was refluxed for 16
hours. The reaction was cooled to about room temperature and
filtered into a larger reactor equipped with a pH electrode
standardized to pH 7-10. The filtered solution was cooled to
5.degree. C. and the product precipitated by slow addition of
dilute acetic acid solution (prepared by mixing glacial acetic acid
(12.6 kg, 210 moles) with 75 kg of water and cooling the mixture to
5.degree. C.) until the pH is between 7.5 and 9.0 (target 8.5). The
resulting slurry was immediately filtered and the filter cake was
recharged back to the reactor. The reactor was charged with 225 kg
of distilled water. The mixture was heated to not more than
50.degree. C. for 30 minutes, then cooled to 15.+-.10.degree. C.
and stirred for 30 minutes. The resulting precipitate was filtered
by vacuum filtration, rinsed with 50 kg of distilled water and
dried in a vacuum oven at not more than 45.degree. C. for not less
than 8 hours to provide the desired product as a tan solid.
c) Preparation of Stearoyl-pivaloyl mixed anhydride
[0413] To 22.4 kg of stearic acid (78.7 moles) in 156.4 kg of
toluene was added 8.2 kg of triethylamine (81.0 moles). The
internal temperature of the resulting slurry was lowered to
-5.degree. C., then 9.52 kg of pivaloyl chloride (79.0 moles) was
slowly added maintaining an internal temperature of not more than
5.degree. C. The slurry was stirred for 2 hours at 5.degree. C.,
then warmed to 20.degree. C. and stirred for 4 hours. The
triethylammonium hydrochloride precipitate was filtered and washed
with 36.6 kg, 35.5 kg and 37.9 kg of toluene. The filtrate was
concentrated at not more than 60.degree. C. internal temperature
and 61.1 kg of heptane was added, followed by cooling the slurry to
-15 to -10.degree. C. After 4 hours of stirring, the resulting
solid was collected by vacuum filtration, blown dry for 1 hour with
nitrogen and dried in a vacuum oven at room temperature for 1.5
hours to provide the desired product as white crystals (18.9 kg). A
further 2.7 kg of the desired product was obtained by concentrating
the mother liquors under vacuum and adding 41.1 kg of heptane. The
resulting slurry was cooled to -15 to -10.degree. C. for 4 hours,
filtered, blown dry with nitrogen for 1 hour and the product dried
in a vacuum oven at room temperature.
d) Preparation of
(R)-9-[4,4-diethoxy-2-(stearoyloxymethyl)butyl]guanine
[0414] The product of Example 30, step b) (3.9 kg, 11.9 moles), the
product of Example 30, step c) (5.2 kg, 13.6 moles) and 300 g of
4-dimethylaminopyridine (2.4 moles) were combined in 103.3 kg of
THF at room temperature. After mixing for 16 hours, water (3 kg)
was added. After mixing for 45 minutes, the solution was distilled
at not more than 45.degree. C. internal temperature. Ethyl acetate
(62.9 kg) was charged and the solution was redistilled at not more
than 45.degree. C. internal temperature. Acetone (56 kg) was then
added and the slurry heated to reflux (56.degree. C.) for 15
minutes. The resulting clear solution was cooled to room
temperature (not more than 15.degree. C./hour). After 4 hours at
room temperature, the resulting precipitate was filtered and rinsed
with acetone (17 kg).
[0415] The mother liquors were concentrated under vacuum at not
more than 45.degree. C. Ethyl acetate (260 kg) and water (72.1 kg)
were charged. The biphasic mixture was stirred and then allowed to
settle. The organic phase was separated and was distilled. Ethyl
acetate (200 kg) was added and the solution was redistilled.
Acetone (101 kg) was charged, the solution heated to reflux
(56.degree. C.) for 15 minutes and then the solution was cooled to
room temperature (not more than 15.degree. C./hour) and the
precipitate was filtered. The product was washed with acetone (19
kg, 15 kg and 15 kg), blown dry with nitrogen for 1 hour and then
dried under vacuum at not more than 40.degree. C. for approximately
6 hours to yield the desired product (3.1 kg).
e) Preparation of
(R)-9-[4-hydroxy-2-(stearoyloxymethyl)butyl]guanine
[0416] The product of Example 30, step d) (3.0 kg) was slurried in
THF (46 L) at 20.degree. C. A solution of trifluoromethanesulfonic
acid (2.25 kg) in 2.25 kg of water (prepared by slowly adding the
acid to cold water) was added and the reaction mixture was stirred
at 22.degree. C. for 2 hours. The reaction mixture was cooled to
15.degree. C. and quenched with a solution of NaHCO.sub.3 (1.5 kg)
in water (5.3 kg). Borane t-butylamine complex (powder, 340 g) was
added in four portions and then the reaction temperature was
increased to 35.degree. C. and stirred for 12 hours. The reaction
mixture was added to a solution of 320 g of concentrated HCl (37%
aq.) in 115 kg of tap water at 5.degree. C. This mixture was
stirred for 30 minutes and the resulting precipitate was filtered
and washed with acetonitrile (15 kg). The solids were
reprecipitated once or twice from acetone (35 kg). A final
precipitation was accomplished by dissolving the product in THF (24
kg) at 65.degree. C., adding water (1.3 kg), cooling to 30.degree.
C. and then adding methylene chloride (105 kg). The resulting
slurry was cooled to 10.degree. C. and the precipitate was filtered
to provide the desired product.
f) Preparation of
(R)-9-[4-(N-benzyloxycarbonyl-L-valyloxy)-2-(stearoyloxy-
methyl)butyl]guanine
[0417] A solution of dicyclohexylcarbodiimide (1500 g, 7.27 moles)
in THF (7 L) was added to a reactor containing a mixture of
N-carbobenzyloxy-L-valine (3630 g, 14.5 moles) in THF (20 L). The
resulting mixture was stirred at 20.+-.5.degree. C. for 1-2 hours.
The product of Example 30, step e) (2500 g, 4.81 moles) and
4-dimethylaminopyridine (59 g, 0.48 moles) were charged to a second
reactor. To this second reactor was filtered the THF mixture from
the first reactor, followed with a rinse of THF (15 L). The
resulting mixture was stirred at 20.+-.5.degree. C. for 1-3 hours.
Water (600 mL) was added and the solution was concentrated under
vacuum at not more than 45.degree. C. The residual oil was taken up
in ethyl acetate (14 L) and filtered. The filtrate was washed
successively with 10% aqueous sodium bicarbonate (2.times.14 L) and
10% brine (14 L). The organic phase was concentrated under vacuum
and the residue was dissolved in methanol (10 kg) at 50-60.degree.
C. The warm solution was added gradually to a mixture of
acetonitrile (30 kg) and water (13 kg) at ambient temperature. The
mixture was stirred 1 hour at 15.degree. C., then filtered to
isolate the crude product, which was dried at 40.degree. C. under
vacuum to provide the desired product as a white solid (3.9
kg).
g) Preparation of
(R)-9-[(2-stearoyloxymethyl)-4-(L-valyloxy)butyl]guanine
[0418] A hydrogenation reactor was charged with 10% Pd--C (400 g)
and the product of Example 30, step f) (2.4 kg). Absolute ethanol
(52 L) was added and the mixture was warmed to 40.degree. C. and
hydrogenated at 30-40 psi for 3-5 hours. On completion of the
reaction, the catalyst was removed by filtration through
diatomaceous earth and the filter cake was rinsed well with ethanol
(30 L). The combined filtrates were concentrated under vacuum at
not more than 60.degree. C. to leave a white solid residue. This
was dissolved in isopropanol (15 L) and isopropyl acetate (60 L) at
reflux and then allowed to cool to room temperature over 4 hours.
After cooling for 3 hours at 15.+-.10.degree. C., the precipitate
was isolated by filtration, washed with isopropyl acetate (6 L) and
dried under vacuum at 40.degree. C. to provide the desired product
as a white powder (864 g).
EXAMPLE 31
Alternative preparation of
(R)-9-[(2-stearoyloxymethyl)-4-(L-valyloxy)buty- l]guanine
a) Preparation of (2R)-4,4-Diethoxy-2-stearoyloxymethyl-butanol
[0419] 24
[0420] Vinyl stearate (17.76 g. 0.057 moles) was charged to a 100
mL round bottom flask with a magnetic stir bar. The flask was
immersed with stirring in a 35.degree. C. oil bath. The product of
Example 14, step b) (10.0 g, 0.052 moles) and Lipase Amano PS-30
(0.20 g) were added and stirred for four hours at 35.degree. C. The
reaction was diluted with hexane (260 mL) and MTBE (115 mL) and
filtered through celite. The filtrate was washed twice with water
(100 mL), dried with Na.sub.2SO.sub.4, and concentrated to provide
the desired product (26.21 g) as a clear oil that forms a wet solid
on standing at room temperature.
b) Preparation of (2S)-4,4-Diethoxy-2-stearoyloxymethyl-butyl
toluenesulfonate
[0421] 25
[0422] The product of Example 31, step a) (26.21 g, 0.057 mol) was
dissolved in methylene chloride (75 mL) and charged into a 250 mL 3
necked flask equipped with a magnetic stir bar, condenser, N.sub.2
inlet, and temperature probe. Triethylamine (14.4 g) was added
followed by p-toluenesulfonyl chloride (16.3 g). The flask was
purged with N.sub.2 and heated to reflux (46.degree. C.). The
reaction was stirred at reflux 6 hours. The reaction was cooled to
room temperature. Water (10 mL) was added and the reaction was
stirred vigorously for 16 hours. The reaction mixture was poured
into a 1 L separatory funnel containing ethyl acetate (350 mL) and
water (350 mL). The organic layer was separated and washed with 7%
(w/w) aq. sodium bicarbonate (100 mL). The organic layer was then
washed with 23% (w/w) aq. sodium chloride (100 mL). The organic
layer was dried with Na.sub.2SO.sub.4 and filtered. The solution
was concentrated to give the desired product (29.4 g) as an oil
that formed a wet solid when cooled to room temperature.
c) Preparation of
(3S)-3-stearoyloxymethyl-4-toluenesulfonyloxy-butyraldeh- yde
[0423] 26
[0424] The product of Example 31, step b) (29.38 g, assayed at
23.12 g, 0.037 moles) was dissolved in THF (90 mL) and charged into
a 250 mL round bottomed flask equipped with a magnetic stir bar and
a temperature probe. Charged water (38 mL) and cooled to 10.degree.
C. Trifluoroacetic acid (55 mL) was poured in and the mixture was
stirred for 25 minutes. The reaction mixture was poured into a 2 L
separatory funnel containing 20% (w/w) K.sub.2CO.sub.3 solution
(690 g), ice (600 g), and ethyl acetate (500 mL). The upper organic
layer was separated. The aqueous layer was extracted a second time
with ethyl acetate (500 mL). The combined organic extracts were
washed with 23% (w/w) NaCl solution. The organic layer was
separated, dried with Na.sub.2SO.sub.4 and filtered. The solution
was concentrated to 21.5 g of an oil, dissolved in heptane (150
mL), and stirred slowly (crystals formed after 10 minutes). The
slurry was stirred 15 hrs. at ambient temperature, filtered and
washed with ambient heptane (20 mL). The desired product was
obtained as white crystals which were dried to a constant weight of
12.3 g.
d) Preparation of
(2S)-4-N-Carbonylbenzyloxy-L-valinyloxy-2-stearoyloxymet- hyl-butyl
toluenesulfonate
[0425] 27
[0426] The product of Example 31, step c) (11.91 g, 0.022 mol) was
charged to a 250 mL shaker bottle. THF (120 mL) and RaNi (17.8 g)
were added. The reaction was pressurized to 4 atm. with H.sub.2.
The reaction was shaken for 1.5 hours. The reaction was filtered
and washed with 20 mL THF. The filtrate is diluted with 100 mL of
CH.sub.2Cl.sub.2, dried with Na.sub.2SO.sub.4, filtered, and washed
with 25 ml CH.sub.2Cl.sub.2. The filtrate was charged to a 500 mL 3
necked flask equipped with a magnetic stir bar and N.sub.2 inlet.
N-Cbz-L-valine (13.88 g, 0.055 moles), 1,3-dicyclohexylcarbodiimide
(11.37 g, 0.055 moles), and 4-dimethylaminopyridine (0.40 g, 0.003
moles) were added and the reaction was stirred for 1 hr. The
reaction mixture became heterogeneous after several minutes. The
reaction was filtered and washed with CH.sub.2Cl.sub.2 (50 mL). The
filtrate was diluted with ethyl acetate (600 mL) and washed twice
with 7% (w/w) NaHCO.sub.3 solution (100 mL). The organic layer was
then washed twice with 5% (w/w) KH.sub.2PO.sub.4 solution (100 mL).
The organic layer was washed with 7% (w/w) NaHCO.sub.3 solution
(100 mL), then dried with MgSO.sub.4 and filtered. The solution was
concentrated to 19.46 g of oily solids. The solid was dissolved in
30 mL of 8:2 hexanes:ethyl acetate and chromatographed in two
parts. Each half was chromatographed on a Flash 40M silica gel
cartridge (90 g of 32-63 .mu.m, 60 .ANG. silica 4.0 cm.times.15.0
cm) and eluted with 8:2 hexanes:ethyl acetate at 25 ml/min. 25 ml
fractions were collected. Fractions were analyzed by TLC. Fractions
10-22 contained pure product in the first run and fractions 9-26
contained pure product in the second run. The fractions were
combined and concentrated to provide the desired product as a clear
viscous oil (12.58 g).
e) Preparation of 6-Benzyloxy-2-amino-purine
[0427] 28
[0428] 60% Sodium hydride in mineral oil (2.36 g, 0.059 moles) was
charged to a 500 mL 3-neck flask equipped with magnetic stirring,
temperature probe, condenser, and N.sub.2 inlet. Toluene (250 mL)
was added. Benzyl alcohol (50 mL) was added dropwise over 30
minutes. After addition of benzyl alcohol, the reaction was stirred
10 minutes. Then 6-chloro-2-aminopurine (5.00 g, 0.029 moles) was
added and the reaction mixture was heated to reflux (115.degree.
C.) for 4.5 hours. The reaction mixture was filtered hot through a
coarse glass fritted funnel and 11.65 g of wet off-white solids
were obtained. The wet solids were triturated with CH.sub.2Cl.sub.2
(100 mL) and water (100 mL). After 10 minutes of stirring the
solids had dissolved. The aqueous layer was separated and the pH
was lowered to 9 over 3 minutes with 6 M HCl. A white solid
precipitate formed. The slurry was filtered, washed with water (50
mL), and dried (in vacuo at 50.degree. C.) to a constant weight to
provide the desired product as off-white crystals (5.15 g).
f) Preparation of
(R)-9-[(2-stearoyloxymethyl)-4-(N-benzyloxycarbonyl-L-va-
lyloxy)butyl]guanine
[0429] 29
[0430] The product of Example 31, step e) (2.40 g, 0.0099 moles)
was charged to a 100 mL round bottom flask equipped with magnetic
stirring and a N.sub.2 inlet. DMF (6 mL) and potassium carbonate
(6.27 g) were added. The mixture was stirred at room temperature
for 30 minutes. The product of Example 31, step d) (7.02 g, 0.0091
moles) was dissolved in DMF (21 mL) and added to the mixture. The
flask was immersed in a 70.degree. C. oil bath and stirred 24
hours. The reaction was cooled to ambient temperature and poured
into a 500 mL separatory funnel containing ethyl acetate (135 mL)
and 5% (w/w) KH.sub.2SO.sub.4 solution (135 mL). The top organic
layer was kept and washed with 7% (w:w) NaHCO.sub.3 solution (100
mL). The organic layer was dried with MgSO.sub.4 and filtered. The
solution was concentrated to 9.79 of oily solids. This was
triturated in 50 mL of 1:1 hexanes:ethyl acetate, filtered, and
concentrated to 9.10 g of yellow oil. The oil was dissolved in 20
mL of 1/1 hexanes-ethyl acetate and chromatographed on a Flash 40M
silica gel cartridge (90 g of 32-63 .mu.m, 60 .ANG. silica, 4.0
cm.times.15.0 cm) eluted with 6:4 hexanes:ethyl acetate at 25
ml/min. 25 ml fractions were collected. Fractions were analyzed by
TLC. Fractions 27-92 contained pure product by TLC. The pure
fractions were combined and concentrated to yield the desired
product as an oil (2.95 g).
g) Preparation of
(R)-9-[(2-stearoyloxymethyl)-4-(L-valyloxy)butyl]guanine
[0431] 30
[0432] The product of Example 31, step f) (2.63 g, 0.0031 moles)
was dissolved in ethanol (50 mL) and charged into a 500 mL round
bottom flask. 10% Pd/C (0.5 g) was slurried in ethanol (20 mL) and
added to the flask. The reaction was stirred under H.sub.2 (1 atm
from balloon) for 1.5 hours. The slurry was heated briefly to
dissolve any solids, filtered through celite, and washed with hot
ethanol (50 mL). The filtrate was concentrated to give 1.752 g of
white solid. The solid was dissolved in isopropyl alcohol (10 mL)
and isopropyl acetate (42 mL) at 70.degree. C. The solution was
cooled to 15.degree. C. over 2 hours and stirred at 15.degree. C.
for 12 hours. The solution was cooled to 0.degree. C. over 30
minutes and stirred for 1 hour. The slurry was filtered and washed
with isopropyl acetate (10 mL). The solid was dried in vacuo at
50.degree. C. to provide the desired product (0.882 g).
[0433] The mother liquors were concentrated to give 0.55 g of white
solid which was dissolved in isopropyl alcohol (3 mL) and isopropyl
acetate (16 mL) at 75.degree. C. The solution was cooled to
15.degree. C. for 2 hours, then filtered and dried as above to to
provide an additional 0.181 g of the desired product.
EXAMPLE 32
Alternative preparation of Ethyl 4,4-Diethoxy-2-ethoxycarbonyl
butyrate
[0434] To a suspension of sodium ethoxide (20 g, 0.294 moles) in
dimethylformamide (68 g) was added diethyl malonate (49 g, 0.306
moles) during 13 minutes. After the addition was complete, the
mixture was heated to 110.degree. C. and bromoacetaldehyde diethyl
acetal (40 g, 0.203 moles) was added over 1 hour and 45 minutes.
After the addition was complete, the mixture was heated at
110.degree. C. for 7 hours. The reaction mixture was cooled to room
temperature and methyl t-butyl ether (160 g) and water (100 g) were
added and the mixture was stirred for 15 minutes. The organic layer
was separated and treated with 7% aqueous potassium hydroxide
solution (155 g). The layers were separated and the organic layer
was washed with water (100 g) and then with brine (60 g). The
organic layer was concentrated to give the crude desired product.
The crude product was heated under house vacuum (approximately 45
mm of Hg) at 160-170.degree. C. (bath temperature) to distill off
the volatile impurities, providing 43.6 g of the desired
product.
EXAMPLE 33
Alternative preparation of
(R)-9-[4,4-diethoxy-2-(hydroxymethyl)butyl]guan- ine
[0435] To a 100 mL one neck flask was added the product of Example
30 a) (5 g, 0.0145 moles), followed by the addition of a solution
of KOH (2.05 g, 0.0445 moles) in water (20 mL). The mixture was
stirred at reflux for 16-20 hours. Then the reaction mixture (at
reflux) was adjusted to pH 7.0 by the addition of acetic acid. The
reaction mixture was then cooled to room temperature and stirred
for 30 minutes. The resulting precipitate was collected by
filtration and washed with water (5 mL). The resulting solid was
dried overnight at not more than 50.degree. C. to provide 4.45 g of
the desired product.
EXAMPLE 34
Alternative purification of
(R)-9-[4-hydroxy)-2-(stearoyloxymethyl)butyl]g- uanine as the
(S)-(+)-camphorsulfonic acid salt
[0436] In a 250 mL round bottom flask was placed the product of
Example 14 i) (13.0 g) and (1S)-(+)-10-camphorsulfonic acid (5.85
g). Heptane (50 mL) was added and the mixture was stirred for 15
minutes. Then tetrahyrofuran (THF; 50 mL) was added and the mixture
was stirred for 5 hours. The resulting precipitate was collected by
filtration and washed with heptane (100 mL). The resulting solid
was dried under vacuum at 45.degree. C. to provide the desired
product (11.3 g). HPLC analysis of the product indicated 98.76%
e.e.
EXAMPLE 35
[0437] Preparation of 31
[0438] A 50 mL round bottom flask was charged with the product of
Example 14 h) (1.0 g, 1.7 mmol), THF (20 mL), H.sub.2O (1 mL), and
Amberlyst 15 resin (1.0 g). The solution was then heated to
65.degree. C. for 3 hours. The solution was then filtered hot and
the resin was washed with THF (2.times.10 mL). The solvent was then
removed under vacuum to give the desired product (0.74 g, 84%).
EXAMPLE 36
Alternative preparation of
(R)-9-[4-hydroxy)-2-(stearoyloxymethyl)butyl]gu- anine
[0439] A 100 mL round bottom flask was charged with the product of
Example 14 h) (2.45 g, 4.14 mmol), THF (25 mL), H.sub.2O (1 mL) and
Amberlyst 15 resin (2.5 g). The solution was then heated to
65.degree. C. for 3 hours. The solution was then filtered hot and
the resin was washed with THF (2.times.15 mL). The solution of the
crude aldehyde was cooled to room temperature and a solution of
borane t-butylamine complex (0.3 g, 3.45 mmol), in THF/H.sub.2O
(1/1 20 mL) was added dropwise to the aldehyde solution. The
solution was stirred at room temperature for 1.5 hours, and the
reaction was then quenched by addition of H.sub.2O (100 mL). After
stirring at room temperature for an additional 30 min., the
precipitate was isolated by filtration and dried to give 1.00 g
(47%) of the desired product.
EXAMPLE 37
Alternative preparation of
(R)-9-[4-(N-benzyloxycarbonyl-L-valyloxy)-2-(st-
earoyloxymethyl)butyl]guanine
a) N-Carbobenzyloxy-L-valine Anhydride
[0440] A solution of dicyclohexylcarbodiimide (5 kg, 24 moles) in
acetonitrile (17.5 kg) was added to a reactor containing a solution
of N-carbobenzyloxy-L-valine (12.5 kg, 50 moles) in acetonitrile
(200 kg). The mixture was stirred at 5+/-5.degree. C. for 6 hours
and the resulting solid was filtered off. The filtrate was
concentrated under vacuum at not more then 45.degree. C. and the
residue was dissolved in toluene (50 kg) at 40.degree. C. Heptane
(50 kg) was added and the mixture was cooled to 15+/-5.degree. C.
The precipitate was filtered off and dried to give 10.2 kg of the
desired product.
b)
(R)-9-[4-(N-benzyloxycarbonyl-L-valyloxy)-2-(stearoyloxymethyl)butyl]-g-
uanine
[0441] A mixture of
(R)-(-[4-hydroxy-2-(stearoyloxymethyl)butyl]guanine (5.2 kg, 10
moles), N-CBZ-L-valine anhydride (6.3 kg, 13 moles),
4-dimethylaminopyridine (60 g, 0.5 moles) and tetrahydrofuran (67
kg) was stirred for 2-4 hours at 25+/-5.degree. C. Water (2 kg) was
added and the mixture was concentrated under vacuum at not more
then 45.degree. C. The residue was dissolved in ethyl acetate (58
kg) and extracted with 10% aqueous sodium bicarbonate (2.times.50
kg) and water (1.times.50 kg). The ethyl acetate solution was
concentrated under vacuum and the residue was dissolved in methanol
(20 kg) at 50+/-5.degree. C. The solution was cooled to
20+/-5.degree. C. and diluted with acetonitrile (50 kg) and water
(3 kg). The precipitate was filtered off and dried under vacuum to
give the desired product (5.3 kg).
EXAMPLE 38
Alternative preparation of
(R)-9-[4,4-diethoxy-2-(stearoyloxymethyl)butyl]- -guanine
[0442] To a stirred solution of stearic acid (1.05 g) and
N-mehtylmorpholine (0.62 g) in THF (13 mL) at 0-4.degree. C. was
added a solution of p-tosyl chloride (0.67 g) in THF (2 mL) at -3
to -4.degree. C. The mixture was stirred at room temperature for 3
hours. The product of Example 14 g) (1.0 g) and
4-dimethylaminopyridine (75 mg) were added and the slurry was
stirred at room temperature for 5 days and quenched with 135 mL of
water. The mixture was stirred overnight and the precipitate was
filtered and washed with water. The wet filter cake was dried under
vacuum (40.degree. C.) to give the desired product (1.3 g) as a
light yellow powder.
EXAMPLE 39
Alternative preparation of
(R)-9-[4,4-diethoxy-2-(hydroxymethyl)butyl]guan- ine
[0443] The product of Example 30 a) (10.0 g, 29.1 mmoles) was added
to a solution of sodium hydroxide (2.33 g, 5.82 mmoles) in water
(200 mL). A solution of trimethylamine (6.61 mL of 40 wt. %
solution in water, 43.6 mmoles) was charged to the suspension. The
heterogeneous mixture was stirred at room temperature overnight.
The reaction was diluted with water (50 mL) and then extracted with
ethyl acetate (200 mL). The water layer was charged with a
saturated solution of ammonium sulfate (300 mL). The mixture was
stirred at room temperature for 30 hours and the resulting
precipitate was filtered. The filter cake was washed with ethyl
acetate (100 mL). The product was dried in a vacuum oven (high
house vacuum, 45.degree. C.) overnight to provide the desired
product (7.88 g).
EXAMPLE 40
Alternative preparation of
(R)-9-[4,4-diethoxy-2-(hydroxymethyl)-butyl]gua- nine
[0444] A 50 gallon stainless steel reactor was purged with nitrogen
and charged with the product of Example 30 a) (13.5 kg) and DMAP
(0.48 kg). To the solids was added methyl t-butyl ether (108 kg),
followed by triethylamine (4.0 kg). Acetic anhydride (4.64 kg) was
added last. The resulting mixture was stirred at ambient
temperature for 30 minutes. Distilled water (56 kg) was charged to
the reactor and the contents were stirred for 30 minutes. After
allowing the mixture to settle for 30 minutes, the lower layer was
drained and 50 kg of saturated brine was added to the reactor. The
contents of the reactor were stirred for 30 minutes and let settle
for 30 minutes. The lower layer was drained and a Karl Fischer
reading was done on the organic layer to assure that the water
content was less than 2.5%. The organic layer was stirred at
ambient temperature for 24 hours. The resulting precipitate was
filtered off and the filtrate was concentrated under vacuum,
followed by a methanol (22 kg) chase. To the resulting residue was
added methanol (49 kg) and 10.8 kg of a 50% aqueous KOH solution.
The mixture was heated to relux for one hour. The methanol was
removed by distillation and the distillation residue was diluted
with distilled water (112 kg) and 9.2 kg of a 50% aqueous KOH
solution. The resulting mixture was heated to reflux for 16 hours.
The contents of the reactor were cooled to 25.degree. C. and were
then adjusted to pH 7.0 using 37% aqueous acetic acid solution. The
internal temperature of the reactor was then adjusted to 10.degree.
C. and the contents stirred for 30 minutes. The resulting slurry
was centrifuged and the resulting wet cake was charged back to the
reactor. To the cake was charged distilled water (70 kg). The
internal temperature was adjusted to 50.degree. C. and the contents
were stirred for 30 minutes. Then the internal temperature was
adjusted to 20.degree. C. and the contents stirred for 30 minutes.
The resulting slurry was centrifuged and the cake rinsed once with
distilled water (15 kg). The cake was transferred to dryer trays
and dried at 45.degree. C. under vacuum for 18 hours to provide the
desired product as a pale yellow powder (8.6 kg, 99% ee).
EXAMPLE 41
Alternative preparation of
(R)-9-[4-hydroxy-2-(stearoyloxymethyl)butyl]-gu- anine
[0445] To a 2 liter round bottom, 3-neck flask equipped with a
nitrogen inlet, temperature probe, rubber septum and mechanical
stirrer was charged stearic acid (25.0 g), THF (525 mL) and
triethylamine (12.2 mL). The resulting solution was cooled to
.sup.30.degree. C. using an ice/salt bath. Pivaloyl chloride (10.3
mL) was added slowly via a syringe, maintaining the reaction
temperature at less than 5.degree. C. The resulting slurry was
stirred at 0.+-.5.degree. C. for 2 hours. The ice bath was removed
and the reaction allowed to warm to room temperature. The resulting
precipitate was filtered and the filter cake was rinsed with THF
(100 mL). The resulting clear filtrate was added to a 3 liter
3-neck flask (equipped with a nitrogen inlet and mechanical
stirrer) charged with the product of Example 40 (22.5 g) and DMAP
(1.7 g). The reaction mixture was stirred overnight at room
temperature. The reaction mixture was then cooled to 18.degree. C.
and a room temperature solution of 1:1 aqueous triflic acid (27.5 g
triflic acid) was added slowly, maintaining the temperature at less
than 23.degree. C. The resulting solution was stirred at
approximately 22 C for 4.5 hours. Then the reaction mixture was
cooled to 18.degree. C. and diulted with water (70 mL). Sodium
bicarbonate was added to adjust the pH to 6-7 (target 6.5). The
mixture was stirred at room temperature for 30 minutes.
[0446] The bath temperature was set at 35.degree. C. and the
borane-t-butylamine complex (4.52 g) was added in several portions
over 50 minutes. The reaction mixture was stirred at 35.degree. C.
overnight. An additional portion of borane-t-butylamine (200 mg)
was added and the mixture stirred for an additional 3 hours. The
reaction mixture was quenched by pouring it into a cold solution of
5 mL of HCL in 625 mL of water. The resulting pH was 5-6 (target
less than pH 6). The resulting mixture was stirred for 3 hours at
room temperature and then filtered. The filter cake was dried
overnight under house vacuum at 35.degree. C. The filter cake,
optionally, can be washed with acetonitrile prior to drying. The
dried solid was suspended in acetone (1100 mL) and heated to
reflux. The slurry was held at reflux for 30 minutes and then
cooled to room temperature. After stirring at room temperature for
one hour, the mixture was filtered. The filter cake was air-dried
on the filter funnel for 30 minutes and then suspended in THF (350
mL). The THF mixture was heated to reflux and water (35 mL) was
added. The flask containing the mixture was removed from the
heating bath and allowed to cool. When the temperature reached less
than 30.degree. C., ethyl acetate (1050 ml) was added and the
mixture was stirred for one hour at room temperature. The resulting
slurry was filtered and the filter cake was dried overnight at
35.degree. C. to provide the desired product as a white powder
(30.4 g).
Example 42
Alternative preparation of
(2S)-4-N-Carbonylbenzyloxy-L-valinyloxy-2-stear- oyloxymethyl-butyl
toluenesulfonate
[0447] The product of Example 31 c) (6.00 g) was dissolved in THF
(60 mL). Borane t-butylamine comlex (0.48 g) was added neat at room
temperature. The reaction mixture was stirred at room temperature
for 1.25 hours. The pH was adjusted to 7-8 by addition of 5%
aqueous HCl. The reaction mixture was diluted with THF (60 mL) and
was washed with 20% brine (40 mL) and then again with saturated
brine (30 mL). The organic solution was filtered through a pad of
silica gel, dried over magnesium sulfate (6.0 g) for one hour and
filtered. The filtrate was added to the product of Example 37 a)
(7.0 g) and DMAP (70 mg). The mixture was stirred under nitrogen at
room temperature for about 3 hours. An additional amount of the
product of Example 37 a) (0.5 g) was added and the mixture was
stirred overnight at room temperature. An additional amount of the
product of Example 37 a) (0.5 g) was added and the mixture was
stirred overnight. The reaction mixture was diluted with ethyl
acetate (90 mL) and washed with half-saturated sodium bicarbonate
(90 mL), with brine (60 mL), with 5% KH.sub.2PO.sub.4 (60 mL) and
brine (60 mL). The organic solution was dried over sodium sulfate
and concentrated to provide the desired product as a yellow oil
(6.88 g).
Example 43
(R)-2-Amino-6-chloro-9-[4-(N-benzyloxycarbonyl-L-valyloxy)-2-(stearoyloxym-
ethyl)butyl]purine
[0448] A 100 ml round bott0m 3-neck flask was charged with lithium
hydride (58 mg, 7.3 mmol) and DMF (10 mL). 2-Amino-6-chloropurine
(1.14 g, 6.72 mmol) was added al at once at room temperature. The
mixture was stirred at room temperature for 40 minutes under
nitrogen. The product of Example 31 d) (5.2 g, 6.72 mmol) as a
solution in DMF (10 mL) was added dropwise. After complete
addition, the reaction mixture was stirred at 40-50.degree. C.
under nitrogen for 27 hours. The reaction mixture was cooled to
room temperature and poured into a separatory funnel containing
ethyl acetate (100 mL) and 5% aqueous KH.sub.2PO.sub.4 (100 mL).
The organic layer was separated and washed with saturated aqueous
sodium bicarbonate (50 mL) and brine (50 mL). The organic phase was
concentrated under vacuum. The crude product was dissolved in
methylene chloride (5 mL) and chromatographed on flash silica gel
(10 g) (eluent: 1% methanol/methylene chloride (1000 mL), 5%
methanol/methylene chloride (250 mL)) to provide the desired
product (3.06 g).
Example 44
Alternative preparation of
(R)-9-[4-(N-benzyloxycarbonyl-L-valyloxy)-2-(st-
earoyloxymethyl)butyl]guanine
[0449] A 25 mL round bottom flask was charged with the product of
Example 43 (0.2 g, 0.26 mol), triethylamine (0.20 mL of 40% aq.
solution), THF (4 mL) and water (1 mL). The resulting solution was
stirred at room temperature for 20 hours. The solvent was removed
under vacuum and the residue was dissolved in ethyl acetate (20
mL). This solution was dried over sodium sulfate and the solvent
was evaporated under vacuum. The crude product was chromatographed
on flash silica gel (10 g) (eluant: 1/10 methanol/methylene
chloride (400 mL)) to give the desired product as a colorless oil
(0.15 g).
Example 45
Alternative preparation of
(R)-9-[4-(N-benzyloxycarbonyl-L-valyloxy)-2-(st-
earoyloxymethyl)butyl]guanine
[0450] The product of Example 43 (145 mg, 0.188 mol) was dissolved
in glacial acetic acid (1.9 mL) and the solution was heated to
110.degree. C. for 3 hours. The solution was then cooled to room
temperature and the acetic acid was removed by distillation under
reduced pressure. The residue was dissolved in ethyl acetate and
washed with water, aqueous sodium bicarbonate and bringe. The
organic solution was evaporated under reduced pressure to give the
desired product (134 mg).
Example 46
Alternative preparation of
(R)-2-Amino-6-chloro-9-[4,4-diethoxy-2-(hydroxy-
methyl)butyl]purine
[0451] DBU (36.8 g, 0.24 mol) was added to a suspension of
2-amino-6-chloropurine (41 g, 0.24 mol) in DMF (340 mL) at room
temperature under nitrogen. After 5 minutes, the product of Example
14 d) (85 g, 0.22 mol) was added. The mixture was stirred at
40-45.degree. C. for 15-20 hours. Then the mixture was diluted with
methyl t-butyl ether (340 mL), toluene (340 mL), water (340 mL) and
brine (340 mL). After mixing for 15 minutes, the organic layer was
separated and the aqueous layer was extracted with toluene
(2.times.300 mL). The combined organic layer was washed with water
(500 mL) and concentrated under vacuum at 60.degree. C. bath
temperature. The resulting oil was diluted with methanol (260 mL)
and cooled to 5.degree. C. A solution of K.sub.2CO.sub.3 (16 g,
0.12 mol) in water (65 mL) was added over 15 minutes maintaining
the reaction mixture temperature below 10.degree. C. The mixture
was stirred at 10.degree. C. for 1 hour. Then the mixture was
diluted with brine (500 mL) and stirred for 30 minutes. The
resulting solid was filtered, washed with 5% methanol in water (50
mL) and the filter cake was dried to give the desired product as a
white solid (39 g).
Example 47
Alternative preparation of
(R)-2-Amino-6-chloro-9-[4,4-diethoxy-2-(acetoxy-
methyl)butyl]purine
[0452] 2-Amino-6-chloropurine (0.6 g, 3.6 mmol) and
tert-butylimino-tri(pyrrolidino)phosphorane (1.1 g, 3.6 mmol) were
mixed in anhydrous THF (4 mL) for 10 minutes at 40.degree. C. The
product of Example 14 d) (1.16 g, 3.0 mmol) was added and the
mixture was stirred at 41-43.degree. C. overnight. The THF was
removed by evaporation under vacuum and the residue was diluted
with methyl t-butyl ether (10 mL), water (5 mL) and brine (5 mL).
The organic layer was separated and the aqueous layer was extracted
with toluene (2.times.10 mL). The combined organic layer was washed
with water (25 mL) and concentrated under vacuum. The residue was
slurried with methyl t-butyl ether (12 mL) and water (0.1 mL) and
filtered. The filtrate was concentrated under vacuum and slurried
with hexane (10 mL) and methyl t-butyl ether (1 mL). The resulting
solid was filtered and dried to provide the desired product (0.73
g).
Example 48
Alternate preparation of
(R)-2-Amino-6-chloro-9-[4-(N-benzyloxycarbonyl-L--
valyloxy)-2-(stearoyloxymethyl)butyl]purine
[0453] The title compound was prepared following the procedure of
Example 47, but substituting the product of Example 31 d) for the
product of Example 14 d).
Example 49
Alternate preparation of
(R)-2-Amino-6-chloro-9-[4-(N-benzyloxycarbonyl-L--
valyloxy)-2-(stearoyloxymethyl)butyl]purine
[0454] The title compound can be prepared following the procedure
of Example 48, but substituting DBU for
tert-butylimino-tri(pyrrolidino)-pho- sphorane.
Example 50
2-Amino-6-iodopurine
[0455] To a 2 liter single-neck round bottom flask with a
mechanical stirrer was charged 2-amino-6-chloropurine (41.0 g, 242
mmol). The flask was cooled in an ice-water bath. The the reaction
flask was charged HI (47% solution, pre-cooled in a refrigerator,
250 mL) in one portion. The resulting suspension was stirred for 16
hours at ice-water bath temperature. Water (500 mL) was charged to
the reaction flask. The suspension was stirred at 0.degree. C. for
1 hour. The precipitate was filtered and washed with water
(3.times.250 mL). The filter cake was transferred to a 250 mL
filtration flask. 6 M NaOH solution (85 mL) was added to the solid
through the filter to rinse out residual solid and wash into the
filter flask. The solution obtained was added slowly to a boiling
solution of acetic acid (25 mL) and water (250 mL). The resulting
suspension was cooled to room temperature and stirred at room
temperature for 2 hours. The solid was collected by centrifugation,
washed with water (2.times.250 mL), followed by heptane (250 mL).
The solid was first spin-dried on the centrifuge for 30 minutes and
then dried in a vacuum oven overnight to provide the desired
product (61.3 g).
Example 51
Alternative preparation of
(R)-9-[4-(N-benzyloxycarbonyl-L-valyloxy)-2-(st-
earoyloxymethyl)butyl]guanine
a)
(R)-2-Amino-6-iodo-9-[4-(N-benzyloxycarbonyl-L-valyloxy)-2-(stearoyloxy-
methyl)butyl]purine
[0456] To a 50 mL single neck round bottom flask was charged the
product of Example 31 d) (2.0 g, 2.58 mmol), 2-amino-6-iodopurine
(0.742 g, 2.84 mmol), DBU (o.425 mL) and DMF (10 mL). The reaction
mixture was stirred for 20 hours at 40.degree. C. Ethyl acetate (30
mL) was added to the reaction mixture and stirring continued for 30
minutes. The reaction mixture was filtered and the filtered solid
was washed with ethyl acetate (2.times.30 mL). The filtrate and
washings were combined and washed with water (3.times.25 mL). The
organic solution was evaporated under vacuum. The residue was
redissolved in ethyl acetate (50 mL) and again evaporated under
vacuum to azeotropically remove any residual water, providing the
desired product (2.1 g).
[0457] .sup.1H NMR (300 MHz, d.sub.6-DMSO): .delta. 8.06 (s, 1H),
7.36 (br s, 5H), 6.78 (br s, 2H) 3.85-4.2 (m, 9H), 2.15 (t, 2H),
0.8-1.7 (m, 43H)
[0458] Mass Spec. (ESI): 863 (M+H).sup.+
b) Alternative preparation of
(R)-2-Amino-6-iodo-9-[4-(N-benzyloxycarbonyl-
-L-valyloxy)-2-(stearoyloxymethyl)butyl]purine
[0459] The desired product was obtained following the procedure of
Example 51 a) with the replacement of DBU by K.sub.2CO.sub.3 (1.5
g).
c)
(R)-9-[4-(N-benzyloxycarbonyl-L-valyloxy)-2-(stearoyloxymethyl)butyl]-g-
uanine
[0460] The product of Example 51 a) (3.4 g, 3.94 mmol),
acetonitrile (45 mL), water (35 mL), acetic acid (45 mL) and sodium
acetate (3.05 g) were mixed and heated to reflux (86-87.degree. C.)
for 30 hours. The volatile solvent was revoed by evaporation under
reduced pressure. The aqueous layer was extracted with ethyl
acetate (3.times.200 mL). The combined extracts were mixed with
saturated sodium bicarbonate (2.times.100 mL) for 30 minutes. The
organic layers were separated and washed with saturated sodium
bicarbonate (100 mL), followed by water washes (3.times.100 mL).
The organic solvent was evaporated under reduced pressure. To the
residue was added anhydrous ethyl acetate (3.times.200 mL), with
evaporation of the solvent each time under reduced pressure, to
provide a solid. The solid was recrystallized from refluxing
acetonitrile (50 mL). After cooling the acetonitrile mixture to
room temperature, it was allowed to stand at room temperature
overnight and then was cooled to -13.degree. C. for 30 minutes. The
resulting solid was collected by filtration, washed with
acetonitrile (2.times.10 mL) and dired in a vacuum oven to provide
the desired product (2.4 g).
Example 52
(R)-2-Amino-6-iodo-9-[4,4-diethoxy-2-(acetoxymethyl)butyl]purine
[0461] To a 100 mL single neck round bottom flask was charged the
product of Example 14 d) (9.3 g, 23.9 mmol), 2-amino-6-iodopurine
(4.8 g, 18.4 mmol), DBU (3.6 mL, 24.0 mmol) and DMF (50 mL). The
mixture was stirred for 16 hours at 45.degree. C. The reaction
mixture was cooled to room temperature and ethyl acetate (250 mL)
was added and stirring continued for 30 minutes. The reaction
mixture was filtered and the filtered solid was washed with ethyl
acetate (2.times.125 mL). The filtrate and washings were combined
and washed with water (4.times.50 mL). The organic solution was
evaporated under reduced pressure. Ethyl acetate (50 mL) was added
to the residue and evaporated under reduced pressure. Methyl
t-butyl ether (300 mL) was added to the residue and stirred. The
resulting solid was filtered and dried to provide the desired
product (8.8 g).
[0462] (K.sub.2CO.sub.3 can be used in place of DBU in the above
procedure to provide the desired product).
[0463] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.81 (s, 1H),
5.12 (br s, 2H), 4.61 (t, 1H), 4.16 (m, 1H), 4.04 (m, 2H), 3.62 (m,
2H), 3.48 (m, 2H), 2.52 (m, 1H), 2.03 (s, 3H), 1.79 (s, 1H), 1.69
(m, 2H), 1.19 (m, 6H).
Example 53
Alternative preparation of
(R)-9-[(2-stearoyloxymethyl)-4-(L-valyloxy)buty- l]-guanine
a) Preparation of
(R)-9-[4-(N-benzyloxycarbonyl-L-valyloxy)-2-(stearoyloxy-
methyl)butyl]-guanine
[0464] To a 500 mL round bottom flask was added the product of
Example 30 e) (10.4 g, 20.0 mmol), the product of Example 37 a)
(11.7 g, 24.2 mmol), DMAP (52 mg, 0.43 mmol) and THF (170 mL). The
mixture was stirred at room temperature for 4 hours. Water (10 mL)
was added and the solvent was evaporated under reduced pressure
(bath temperature of approximately 45.degree. C.). Residual THF was
chased with ethyl acetate (40 mL). The residue was dissolved in
ethyl acetate (200 mL) and the solution was washed with saturated
sodium bicarbonate (3.times.100 mL) and then water (100 mL) and the
organic solution was evaporated under reduced pressure (bath
temperature of approximately 45.degree. C.). Residual ethyl acetate
was chased with isopropanol (25 mL) to provide the desired product
in crude form as 14 g of an orange, sticky solid.
b) Preparation of
(R)-9-[(2-stearoyloxymethyl)-4-(L-valyloxy)butyl]-guanin- e
[0465] To the flask containing the crude product of Example 53 a)
was added isopropanol/THF (4/1, 100 mL) and the mixture was heated
to 45-50.degree. C. to dissolve the solids. The solution was cooled
to room temperature. To a separate 500 mL round bottom flask was
added 10% Pd/C (1.00 g) and the flask was evacuated and back-filled
with nitrogen three times. Then isopropanol/THF (4/1, 25 mL) was
added. The solution of the product of Example 53 a) was then added
to the catalyst flask, along with two 35 mL isopropanol/THF (4/1)
rinses. The reaction flask was then evacuated and back-filled with
hydrogen three times. The solution was then heated to 40-45.degree.
C. for 16 hours. Then the hydrogen-filled balloon was replaced with
a condenser and the reaction mixture was heated to 65.degree. C.
for 25 minutes. The reaction mixture was then filtered through
celite (6.05 g) and the filter cake was washed with isopropanol/THF
(4/1, 2.times.50 mL). The filtrate was concentrated under vacuum
(bath temperature 45.degree. C.) and residual THF was chased with
isopropanol (50 mL).
[0466] To the flask was added isopropanol (50 mL) and the mixture
was heated to about 80.degree. C. to dissolve the solids. Isopropyl
acetate (150 mL) was added and heating was continued to dissolve
the solid which formed. Once all solids were dissolved, the
solution was cooled to room temperature and stirred for 12 hours.
The resulting solid was filtered and dried to provide a light gray
solid (9.0 g). This solid was added to a 500 mL round bottom flask,
along with activated carbon (2.25 g) and isopropanol (200 mL). The
mixture was heated to 60-65.degree. C. for 1 hour and then filtered
through celite (6.00 g). The celite cake was washed with hot
isopropanol (65.degree. C., 2.times.50 mL) and the filtrate was
concentrated under reduced pressure (bath temperature of 50.degree.
C.). Isopropanol (40 mL) was added to the residue and the mixture
was heated to 80.degree. C. to dissolve the solids. Isopropyl
acetate (120 mL) was added and heating was continued to dissolve
the precipitate which formed. The solution was cooled to room
temperature and stirred for 12 hours. The resulting solid was
filtered and dried to give the desired product as a white solid
(7.7 g).
[0467] Alternatively, the crude product of the hydrogenation
reaction was mixed with isopropanol (50 mL) and the mixture was
heated to 65-70.degree. C. to dissolve the solids. Acetonitrile (65
mL) was added dropwise via an addition funnel at a rate to
maintain, the temperature above 55.degree. C. During addition of
the acetonitrile, a fluffy gray precipitate formed. After addition
of the acetonitrile was complete, the mixture was heated at
65.degree. C. for 30 minutes and then filtered through a pad of
celite in a steam jacketed funnel. The filtrate was concentrated
and residual acetonitrile was chased with isopropanol (70 mL). The
resulting solid was recrystallized from isopropanol/isopropyl
acetate (30/90 mL) and after stirring at room temperature for 6
hours, the solid was filtered and dried to give the desired product
as a white solid (6.72 g).
Example 54
Alternative preparation of
(R)-9-[4,4-diethoxy-2-(hydroxymethyl)butyl]-gua- nine
a)
2-N-Acetyl-6-O-diphenylcarbamoyl-(R)-9-[4,4-diethoxy-2-(hydroxymethyl)b-
utyl]-guanine
[0468] To a 50 mL round bottom flask was added
2-N-acetyl-6-O-diphenylcarb- amoylguanine (1.10 g, 2.83 mmol) and
anhydrous DMF (10 mL). DBU (423 .mu.L, 2.83 mmol) was added and the
solid dissolved after stirring for 5 minutes. A solution of the
product of Example 14 d) (1.0 g, 2.6 mmol) in anhydrous DMF (5.0
mL) was added and the resulting solution was stirred at 45.degree.
C. under nitrogen for 28 hours. After cooling to room temperature,
the reaction mixture was diluted with ethyl acetate (40 mL) and
water (20 mL). The organic layer was separated and washed with a 5%
KHSO.sub.4 solution, a saturated sodium bicarbonate solution and
brine and then dried over sodium sulfate. The solvent was
evaporated under vacuum to provide a light yellow oil, which was
chromatographed on silica gel (5% heptane in ethyl acetate) to
provide the desired product as a light yellow solid (460 mg).
[0469] .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.--1.05-1.18 (m, 6H),
1.55-1.68 (m, 2H), 1.92 (s, 3H), 2.40-2.52 (m, 1H), 2.47 (s, 3H),
3.32-3.46 (m, 2H), 3.48-3.62 (m, 2H), 3.89-4.02 (m, 2H), 4.10-4.25
(m, 2H), 4.52 (t, J=5.4 Hz, 1H), 7.05-7.42 (m, 10H), 7.91 (s, 1H),
8.11 (s, 1H)
[0470] ESI (-) MS m/z 603 (M-H).sup.-.
b) (R)-9-[4,4-diethoxy-2-(hydroxymethyl)butyl]-guanine
[0471] To the product of Example 54 a) (100 mg, 0.165 mmol) in a 25
mL round bottom flask was added KOH (62 mg, 0.972 mmol) and water
(10 mL). The suspension was refluxed for 20 hours. The reaction
mixture was cooled to room temperature and acidified to pH 5 using
acetic acid. The solvent was evaporated under reduced pressure to
provide the desired product as a white solid.
Example 55
2-N-Acetyl-(R)-9-[4,4-diethoxy-2-(hydroxymethyl)butyl]-guanine
[0472] To a 50 mL round bottom flask was added 2-N-acetyl-guanine
(547 mg g, 2.83 mmol) and the product of Example 14 d) (1.0 g, 2.6
mmol). Anhydrous DMSO (10 mL) was added, folowed by DBU (430 .mu.L,
2.88 mmol). The resulting solution was stirred at 40.degree. C.
under nitrogen for 24 hours. After cooling to room temperature, the
reaction mixture was diluted with chloroform (50 mL) and water (20
mL). The organic layer was separated and washed with water
(2.times.) and brine and then dried over sodium sulfate. The
solvent was evaporated under vacuum to provide a light yellow oil,
which was chromatographed on silica gel (10% methanol in ethyl
acetate) to provide the desired product as a white foam (280
mg).
[0473] .sup.1H NMR (300 MHz, CDCl.sub.3) .sub.--1.10-1.31 (m, 6H),
1.62-1.85 (m, 2H), 2.06 (s, 3H), 2.44 (s, 3H), 2.50-2.68 (m, 1H),
3.40-3.56 (m, 2H), 3.57-3.73 (m, 2H), 3.96-4.20 (m, 2H), 4.32-4.55
(m, 2H), 4.62 (t, J=5.5 Hz, 1H), 7.82 (s, 1H), 11.60 (s, 1H), 12.40
(s, 1H).
Example 56
Alternative preparation of
(R)-9-[(2-stearoyloxymethyl)-4-(L-valyloxy)buty- l]-guanine
[0474] To a 500 ml 3-neck round bottom flask equipped with a
magnetic stirrer and a temperature probe was added the product of
Example 30f) (5.5 g), THF (65 mL) and isopropanol (65 mL). The
clear solution was purged three times with nitrogen and 5%
Pd/BaCO.sub.3 (0.6 g) was added. The mixture was stirred at
40.degree. C. under a hydrogen filled balloon for 16 hours. The
reaction mixture was filtered through celite and the filtrate was
evaporated to dryness to provide a white solid. The solid was
dissolved in isopropanol (25 mL) at 70.degree. C. and isopropyl
acetate (100 mL) was added. The resulting mixture was cooled to
room temperature and stirred for 1 hour. The resulting solid was
filtered and dried under vacuum to provide the desired product as a
white solid (3.39 g).
Example 57
Alternative preparation of 2-Amino-6-benzyloxypurine
[0475] To a 500 mL 3 neck round bottom flask equipped with a
magnetic stirrer, temperature probe and nitrogen inlet was added
2-amino-6-chloropurine (20 g), sodium hyroxide (28 g) and benzyl
alcohol (200 mL). The mixture was stirred for 20 minutes and then
heated at 100.degree. C. for 2-3 hours. The reaction mixture was
then cooled to room temperature and partitioned between methyl
t-butyl ether (300 mL) and water (300 mL). The aqueous layer was
separated and the pH was adjusted to 7-8 with 6 M HCl. The
resulting solid was filtered, washed with water (50 mL) and dried
under vacuum at 50.degree. C. for 20 hours to provide the desired
product as a pale yellow solid (24.3 g).
Example 58
Alternative preparation of
(3S)-3-stearoyloxymethyl-4-toluenesulfonyloxy-b- utyraldehyde
[0476] To a 1 liter 3 neck round bottom flask equipped with a
magnetic stirrer, temperature probe and nitrogen inlet was added
the product of Example 31 b) (40 g) and THF (320 mL). The solution
was cooled to 20.degree. C. and a solution of trifluoromethane
sulfonic acid (20 g) and water (20 g) was added. After stirring for
2-3 hours, the reaction mixture was quenched with sodium
bicarbonate (12.0 g), followed by addition of methyl t-butyl ether
(500 mL). The organic layer was separated and washed with saturated
aqueous sodium bicarbonate solution (200 mL), water (200 mL) and
brine (200 mL) and then was dried over sodium sulfate. The organic
solution was evaporated to dryness under vacuum to give a pale
yellow oil which was dissolved in hexane (300 mL) and stirred
overnight. The resulting solid was filtered and dried under vacuum
to give the desired product as a white solid (25.6 g).
Example 59
Alternative preparation of
(3S)-3-stearoyloxymethyl-4-toluenesulfonyloxy-b- utyraldehyde
[0477] To a 100 mL 3 neck round bottom flask equipped with a
magnetic stirrer, temperature probe and a nitrogen inlet was added
the product of Example 31 b) (6.5 g), acetic acid (30 mL) and
formic acid (20 mL). After stirring at room temperature for 20
minutes, water (20 mL) was added to the mixture and stirring was
continued at room temperature for 30 minutes. The resulting
precipitate was filtered and dried for 1.5 hours. The solid was
added to a 100 mL flask, followed by addition of hexane (90 mL).
The mixture was stirred overnight. The resulting solid was filtered
and dried at 40.degree. C. udner vacuum for 20 hours to provide the
desired product as a white solid (4.6 g).
Example 60
Alternative preparation of N-Carbobenzyloxy-L-valine Anhydride
[0478] A solution of N-Benzyloxycarbonyl-L-valine (20.0 g) in
isopropyl acetate/toluene (1:1. 80 mL) was cooled to 0.degree. C. A
solution of DCC (8.2 g) in toluene (20 mL) was added slowly, at a
rate such that the internal temperature of the reaction mixture was
kept below 10.degree. C. The addition funnel was washed with
toluene (20 mL). The reaction mixture was stirred for 1 hour and
then allowed to warm to room temperature and stirred for another 1
hour. The reaction mixture was filtered and the filter cake was
washed with toluene (20 mL). Heptane (120 mL) was added to the
filtrate and the resulting solution was cooled to 0-5.degree. C.
and stirred for 1 hour. The resulting solid was filtered and washed
with heptane (20 mL) and then dried under vacuum at 35.degree. C.
for 18 hours to provide the desired product as a white solid (17.0
g).
Example 61
Alternative preparation of
(R)-9-[(2-stearoyloxymethyl)-4-(L-valyloxy)buty- l]-guanine
a) Preparation of (2R)-4,4-Diethoxy-2-stearoyloxymethyl-butanol
[0479] Vinyl stearate (3202 g, 9.375 moles) was charged to a 12
liter 4 neck Morton flask with nitrogen inlet and mechanical
stirring. Heating was applied via a 50.degree. C. water bath. As
the vinyl stearate melted, the water bath temperature was decreased
to 35.degree. C. and stirring was started. Heating and stirring was
continued until the vinyl stearate was completely melted. Then the
product of Example 14 b) (1800 g, 9.375 moles) and Lipase PS30 (45
g, 2.5 wt %) were added. The suspension was stirred at
35-37.degree. C. for 22 hours. The reaction mixture was quenched by
additoin of 37.5% methyl t-butyl ether in heptane (2.5 L). The
mixture was then filtered through celite and the celite was washed
with 37.5% methyl t-butyl ether in heptane (12 L). The organic
filtrates were combined and washed with water (10 L) and 23% NaCl
solution (10 L). The organic solution was evaporated and methylene
chloride was aded (4 L). The solution was evaporated to about half
of its original volume. An additional 4 L of methylene chloride was
added and the solution was allowed to stand at 5.degree. C.
overnight:
b) Preparation of (2S)-4,4-Diethoxy-2-stearoyloxymethyl-butyl
toluenesulfonate
[0480] 32
[0481] The methylene chloride product solution resulting from
Example 61 a) was added to a 50 L round bottom flask equipped with
mechanical stirring, water condenser, nitrogen inlet and a
temperature probe. An additional 4 L of methylene chloride was
added, followed by triethylamine (2349 g, 23.2 moles) and
p-toluenesulfonyl chloride (2654 g, 13.92 mol). The reaction
mixture was stirred for 6 hours without external heating or
cooling. Water (1.8 L) was added to the reaction mixture and
stirred vigorously for 17 hours. The organic layer was separated
and washed with water (10 L). The aqueous layer was extracted with
methylene chloride (1 L). The combined organic layers were washed
with 7% sodium bicarbonate solution (10 L) and 23% NaCl solution
(10 L). The solvent was evaporated to provide the desired product
as a thick oil (5947 g).
c) Preparation of
(3S)-3-stearoyloxymethyl-4-toluenesulfonyloxy-butyraldeh- yde
[0482] 33
[0483] A suspension of the product of Example 61 c) (4573 g, 7.47
mol) in acetonitrile (4 L) was added to a 50 L reactor equipped
with a thermocouple and nitrogen inlet. An additional 13 L of
acetonitrile was added and the suspension was heated to 37.degree.
C. with steam. A solution of triflic acid (1253 mL, 14.16 mol) in
water (7.6 L) was added over 20 minutes. Then the mixture was
stirred at 39-42.degree. C. for 1 hour. The reaction mixture was
quenched by adding it to 20 L of 23% aqueous sodium bicarbonate
solution and 35 L of methyl t-butyl ether. The reaction flask was
rinsed with 5 L of methyl t-butyl ether and an additional 20 L of
23% aqueous sodium bicarbonate was addded. This mixture was stirred
for 10 minutes and the layers were separated. The organic layer was
washed with a mixture of 25 L of 23% aqueous sodium bicarbonate
solution and 15 L of 7% NaCl solution. Then the organic layer was
washed with 25 L of 7% NaCl solution. The solvents were removed on
a batch concentrator to provide a thick slurry. Heptane (32 L) was
added to the slurry and then evaporated. Additional heptane (12 L)
was added and evaporated. A further amount of heptane (40 L) was
added and the suspensin was heated to 44.degree. C. in 60 minutes,
causing complete dissolution. The reaction flask was cooled to
40.degree. C. in 10 minutes by running cold water over the surface
of the flask. The solution was then allowed to slowly cool to
35.degree. C., where cyrstallization occurs. The resulting thick
mixture was stirred for 14 hours. The precipitate was filtered and
rinsed twice with 4 L of heptane and then dried on the filter
funnel for 2 hours and then in a vacuum oven with nitrogen purge
for 60 hours at room temperature. The resulting solid (3200 g),
heptane (30 L) and methyl t-butyl ether (1.6 L) were combined and
heated with stirring to dissolution. The resulting solution was
cooled over 1 hour to 42.degree. C. and the resulting suspension
was stirred for 20 hours while cooling to room temperature. The
precipitate was filtered and dried in a vacuum oven with nitrogen
purge for 20 hours at room temperature to give the desired product
(2860 g).
d) Preparation of
(2S)-4-N-Carbonylbenzyloxy-L-valinyloxy-2-stearoyloxymet- hyl-butyl
toluenesulfonate
[0484] 34
[0485] A solution of the product of Example 61 c) (511 g, 950 mmol)
in THF (2.55 L) was stirred at ambient temperature in a
high-pressure reactor with Raney Ni (383 g wet weight) under a 40
psi atmosphere of hydrogen for 2 hours. The suspension was filtered
and the filtrate was swirled with magnesium sulfate (250 g) for 1
hour. The organic solution was filtered and added to N-Cbz-L-valine
anhydride (598 g, 1.23 mol) and DMAP (5.8 g, 47.5 mmol) and stirred
at ambient temperature for 20 hours. The reaction mixture was
poured into 5% KH.sub.2PO.sub.4 (2.5 L) and extracted with methyl
t-butyl ether (2.5 L). The organic layer was washed with 10%
potassium carbonate (2.times.2.5 L) and then 23% NaCl solution (2.5
L). The volatiles were evaporated and methyl t-butyl ether (1 L)
was added. The volatiles were again evaporated and this procedure
repeated (usually about three times) until the Karl-Fischer test
indicated less than 1 mole % water. The organic solution was then
concentrated and stored as an approximately 65% w/w solution of the
desired product.
e) Preparation of
2-Amino-6-iodo-(R)-9-[(2-stearoyloxymethyl)-4-(N-benzylo-
xycarbonyl-L-valyloxy)butyl]purine
[0486] 35
[0487] To a 500 mL flask equipped with a stir bar and a nitrogen
inlet was added
(2S)-4-N-Carbonylbenzyloxy-L-valinyloxy-2-stearoyloxymethyl-butyl
toluenesulfonate (21.8 g, 28.2 mmol), 2-amino-6-iodopurine (9.73 g,
37.3 mmol) and potassium carbonate (11.88 g, 86.1 mmol) slurried in
DMF (155 mL). The resulting mixture was stirred for 16 hours at
50.degree. C. The mixture was then cooled to room temperature and
poured into 400 mL of ethyl acetate and washed with water
(3.times.400 mL). The aqueous washes were combined and extracted
with isopropyl acetate (50 mL). The organic extracts were combined,
washed with brine (200 mL), dried over magnesium sulfate and
concentrated under vacuum. The residue was dissolved in
acetonitrile (150 mL) and washed with heptane. The bottome layer
was separated and concentrated. The residue was dissolved in
methylene chloride (200 mL). Silica gel (60 g) was added and
stirred for 10 minutes. This mixture was poured into a funnel
containing 40 g of silica gel. The product was eluted off of the
silica gel by washing with 4/1 methyl t-butyl ether/heptane. The
filtrate was concentrated to provide the desired product (19.6
g).
f) Preparation of
(R)-9-[(2-stearoyloxymethyl)-4-(N-benzyloxycarbonyl-L-va-
lyloxy)butyl]guanine
[0488] 36
[0489] Into a 300 mL Fisher-Porter bottle (stirbar/nitrogen) was
placed the product of Example 61 e) (12.36 g, 14.34 mmol) dissolved
in acetonitrile (98 mL) and glacial acetic acid (98 mL), followed
by addition of sodium acetate trihydrate (11.70 g, 86 mmol). The
resulting mixture was stirred at 120.degree. C. for 4 hours. The
mixture was cooled to room temperature and poured into 400 mL of
methyl t-butyl ether. The mixture was washed with 5% aq. NaCl
(2.times.300 mL), 2 M potassium carbonate (150 mL), 1% NaHSO.sub.3
(100 mL) and brine (100 mL). The organic layer was concentrated
under vacuum. The residue was dissolved in heptane (150 mL) and
extracted with acetonitrile (2.times.100 mL). The top layer
(heptane) was concentrated to give the desired product as a thick
syrup (8.98 g).
g) Preparation of
(R)-9-[(2-stearoyloxymethyl)-4-(valyloxy)butyl]-guanine
[0490] Into a 100 mL shaker was placed
(R)-9-[(2-stearoyloxymethyl)-4-(N-b-
enzyloxycarbonyl-L-valyloxy)butyl]guanine (4.53 g, 6.03 mmols)
dissolved in isopropanol (45 mL), followed by addition of 4% Pd/C
(450 mg). The resulting mixture was shaken under a 5 psi hydrogen
for 3 days. The mixture was filtered and concentrated under vacuum
to provide a waxy solid. This material was dissolved in hot
isopropanol (12 mL) and isopropyl acetate was added (24 mL). The
mixture was slowly cooled to 40.degree. C. and then stirred at
0.degree. C. for 1 hour. The precipitate was filtered and washed
with isopropyl acetate (5 mL) and then dried to provide the desired
product (1.53 g).
Example 62
Alternative preparation of
(R)-9-[(2-stearoyloxymethyl)-4-(L-valyloxy)buty- l]-guanine
a) Preparation of
(2S)-4-N-t-butyloxycarbonyl-L-valinyloxy-2-stearoyloxyme-
thyl-butyl toluenesulfonate
[0491] 37
[0492] A solution of the product of Example 61 c) (3.10 g, 5.75
mmol) in THF (50 mL) was stirred at ambient temperature in a
high-pressure reactor with Raney Ni (5 g wet weight) under a 5 psi
atmosphere of hydrogen for 3 hours. The suspension was filtered and
the filtrate was swirled with magnesium sulfate (8 g). The organic
solution was filtered and N-Boc-L-valine anhydride (3.11 g, 7.47
mmol) was added, followed by DMAP (0.105 g). The resulting mixture
was stirred at ambient temperature for 30 minutes. The mixture was
cooled to 0.degree. C. and treated with N,N-dimthylethylenediamine
(125 mg). The resulting solution was stirred for 20 minutes and
poured into methyl t-butyl ether (100 mL) and was washed with 5%
KH.sub.2PO.sub.4 (100 mL), 1 M potassium carbonate (100 mL) and
then 27% NaCl solution (20 mL). The organic solution was then
concentrated under vacuum to provide the desired product (3.67
g).
[0493] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 0.88 (m, 6H),
0.95 (d, 3H), 1.25 (m, 30H), 1.45 (s, 9H), 1.55 (m, 2H), 1.70 (m,
2H), 2.1 (m, 1H), 2.21 (t, 2H), 2.46 (s, 3H), 3.94-4.2 (m, 6H), 5.0
(m, 1H), 7.37 (m, 2H), 7.78 (m, 2H). Mass Spec.=740 (M+H).sup.+
b) Preparation of
2-Amino-6-iodo-(R)-9-[(2-stearoyloxymethyl)-4-(N-t-butyl-
oxycarbonyl-L-valyloxy)butyl]purine
[0494] 38
[0495] To a 100 mL flask equipped with a stir bar and a nitrogen
inlet was added the product of Example 62 a) (3.67 g, 4.97 mmol),
2-amino-6-iodopurine (1.68 g, 6.46 mmol) and potassium carbonate
(2.05 g, 14.9 mmol) slurried in DMF (27 mL). The resulting mixture
was stirred for 16 hours at 50.degree. C. The mixture was then
cooled to room temperature and poured into 100 mL of ethyl acetate
and washed with KH.sub.2PO.sub.4 (100 mL containing 20 mL of
brine). The organic phase was washed with brine (2.times.75 mL),
dried over magnesium sulfate, filtered and concentrated under
vacuum. The residue was dissolved in acetonitrile (20 mL) at
50.degree. C. The mixture was cooled to room temperature and
stirred for 2 hours. The precipitate was filtered, washed with
acetonitrile (2.times.5 mL) and dried to provide the desired
product (2.79 g).
[0496] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 0.87 (m, 6H),
0.95 (d, 3H), 1.25 (m, 30H), 1.43 (s, 9H), 1.6 (m, 2H), 1.74 (m,
2H), 2.1 (m, 1H), 2.28 (t, 2H), 2.52 (m, 1H), 4.1-4.4 (m, 6H), 5.03
(m, 1H), 5.22 (s, 1H), 7.73 (s, 1H). Mass Spec.=829 (M+H).sup.+
c) Preparation of
(R)-9-[(2-stearoyloxymethyl)-4-(N-t-butyloxycarbonyl-L-v-
alyloxy)butyl]-guanine
[0497] Into a 4 mL vial (stir bar/nitrogen) was placed the product
of Example 62 b) (0.076 g, 0.092 mmol) dissolved in acetonitrile
(0.444 mL) and glacial acetic acid (0.444 mL), followed by addition
of sodium acetate trihydrate (0.031 g). The resulting mixture was
stirred at 100.degree. C. for 16 hours. HPLC analysis of the
mixture indicated that the desired product had been obtained, by
comparison with authentic product obtained as described in Example
17 b).
d) Preparation of
(R)-9-[(2-stearoyloxymethyl)-4-(valyloxy)butyl]-guanine
[0498] Into a 20 mL vial (stir bar/nitrogen) was added
(R)-9-[(2-stearoyloxymethyl)-4-(N-t-butyloxycarbonyl-L-valyloxy)butyl]-gu-
anine (0.218 g, 0.29 mmol) dissolved in methylene chloride (3.1 mL)
and trifluoroacetic acid (0.33 mL). The resulting mixture was
stirred at 25.degree. C. for 14 hours. The mixture was diluted with
methylene chloride (10 mL), washed with 7% sodium bicarbonate,
dried over magnesium sulfate and concentrated under vacuum to
provide the desired product (161 mg).
Example 63
Alternative preparation of
(R)-9-[(2-stearoyloxymethyl)-4-(L-valyloxy)buty- l]-guanine
a) Preparation of
(2S)-4-N-allyloxycarbonyl-L-valinyloxy-2-stearoyloxymeth- yl-butyl
toluenesulfonate
[0499] 39
[0500] A solution of the product of Example 61 c) (15.0 g, 27.7
mmol) in THF (100 mL) was stirred at ambient temperature in a
high-pressure reactor with Raney Ni (16 g wet weight) under a 5 psi
atmosphere of hydrogen for 3 hours. The suspension was filtered and
the filtrate was swirled with magnesium sulfate (8 g). The organic
solution was filtered and N-Alloc-L-valine anhydride (13.82 g, 43.3
mmol) was added, followed by DMAP (0.203 g). The resulting mixture
was stirred at ambient temperature overnight. The mixture was
diluted with methyl t-butyl ether (120 mL) and was washed with 5%
KH.sub.2PO.sub.4 (25 mL), 1 M potassium carbonate (100 mL) and then
27% NaCl solution (20 mL). The organic solution was then
concentrated under vacuum to provide the desired product (20.6
g).
[0501] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 0.88 (m, 6H),
0.95 (d, 3H), 1.25 (m, 30H), 1.55 (m, 2H), 1.70 (m, 2H), 2.12 (m,
1H), 2.20 (t, 2H), 2.46 (s, 3H), 3.94-4.25 (m, 6H), 4.57 (m, 2H),
5.20-5.35 (m, 3H), 5.90 (m, 1H), 7.45 (m, 2H), 7.79 (m, 2H).
b) Preparation of
2-Amino-6-iodo-(R)-9-[(2-stearoyloxymethyl)-4-(N-allylox-
ycarbonyl-L-valyloxy)butyl]purine
[0502] 40
[0503] To a 500 mL flask equipped with a stir bar and a nitrogen
inlet was added the product of Example 63 a) (18.43 g, 25.4 mmol),
2-amino-6-iodopurine (8.61 g, 33.0 mmol) and potassium carbonate
(10.51 g, 76.2 mmol) slurried in DMF (137 mL). The resulting
mixture was stirred for 16 hours at 50.degree. C. The mixture was
then cooled to room temperature and poured into 394 mL of isopropyl
acetate and washed with water (3.times.400 mL). The organic phase
was washed with brine (200 mL), dried over magnesium sulfate,
filtered and concentrated under vacuum. The residue was dissolved
in acetonitrile (200 mL). The mixture was stirred for 3 hours at
room temperature. The precipitate was filtered, washed with
acetonitrile (2.times.25 mL) and dried to provide the desired
product (12.28 g).
[0504] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 0.89 (m, 6H),
0.98 (d, 3H), 1.29 (m, 30H), 1.6 (m, 2H), 1.74 (m, 2H), 2.13 (m,
1H), 2.28 (t, 2H), 2.52 (m, 1H), 3.9-4.4 (m, 6H), 4.58 (d, 2H),
5.20-5.35 (m, 3H), 5.90 (m, 1H), 7.76 (s, 1H). Ic Mass Spec.=813
(M+H).sup.+
c) Preparation of
(R)-9-[(2-stearoyloxymethyl)-4-(N-allyloxycarbonyl-L-val-
yloxy)butyl]-guanine
[0505] Into a 60 mL sealed tube (stir bar) was placed the product
of Example 63 b) (1.00 g, 1.23 mmol) dissolved in acetonitrile (6.0
mL) and glacial acetic acid (6.0 mL), followed by addition of
sodium acetate trihydrate (1.00 g). The resulting mixture was
stirred at 120.degree. C. for 4 hours. The mixture was cooled to
room temperature and poured into 15 mL of methyl t-butyl ether,
washed with 5% NaCl (2.times.15 mL), 2 M potassium carbonate
(2.times.20 mL), 1% NaHSO.sub.3 (2.times.15 mL) and brine (15 mL).
The organic phase was concentrated under vacuum. The residue was
chromatographed on silica gel (9/1 methylene chloride/methanol) to
provide the desired product as a wax (0.67 g).
[0506] .sup.1H NMR (300 MHz, d.sub.6-DMSO): .delta. 0.85 (m, 9H),
1.21 (m, 30H), 1.45 (m, 2H), 1.62 (m, 2H), 1.99 (m, 1H), 2.22 (t,
2H), 2.35 (m, 1H), 3.8-4.0 (m, 4H), 4.12 (t, 2H), 4.46 (m, 2H),
5.15-5.3 (m, 2H), 5.88 (m, 1H), 6.38 (b s, 2H), 7.63 (s, 1H), 10.52
(b s, 1H). Ic Mass Spec.=703 (M+H).sup.+
d) Preparation of
(R)-9-[(2-stearoyloxymethyl)-4-(valyloxy)butyl]-guanine
[0507] Into a 4 mL vial (stir bar/nitrogen) was added the product
of Example 63 c) (0.07 g, 0.10 mmol) dissolved in THF (1.0 mL) and
triphenylphosphine (1.6 mg) and Pd.sub.2(dba).sub.3 (1.4 mg) and
pyrrolidine (0.071 g). The resulting mixture was stirred at
25.degree. C. for 14 hours. The mixture was concentrated under
vacuum, diluted with isopropanol and stirred at 4.degree. C. The
resulting precipitate was filtered to provide the desired product
(33 mg).
FORMULATION EXAMPLE A
[0508] Tablet Formulation
[0509] The following ingredients are screened through a 0.15 mm
sieve and dry-mixed
[0510] 10 g
(R)-9-[2-(stearoyloxymethyl)-4-(L-valyloxy)butyl]guanine
[0511] 40 g lactose
[0512] 49 g crystalline cellulose
[0513] 1 g magnesium stearate
[0514] A tabletting machine is used to compress the mixture to
tablets containing 250 mg of active ingredient.
FORMULATION EXAMPLE B
[0515] Enteric Coated Tablet
[0516] The tablets of Formulation Example A are spray coated in a
tablet coater with a solution comprising
[0517] 120 g ethyl cellulose
[0518] 30 g propylene glycol
[0519] 10 g sorbitan monooleate
[0520] add 1 000 ml distilled water
FORMULATION EXAMPLE C
[0521] Controlled Release Formulation
[0522] 50 g
(R)-9-[2-(stearoyloxymethyl)-4-(L-valyloxy)butyl]guanine
[0523] 12 g hydroxypropylmethylcellulose (Methocell K15)
[0524] 4.5 g lactose
[0525] are dry-mixed and granulated with an aqueous paste of
povidone. Magnesium stearate (0.5 g) is added and the mixture
compressed in a tabletting machine to 13 mm diameter tablets
containing 500 mg active agent.
FORMULATION EXAMPLE D
[0526] Soft Capsules
[0527] 250 g
(R)-9-[2-(stearoyloxymethyl)-4-(L-valyloxy)butyl]guanine
[0528] 100 g lecithin
[0529] 100 g arachis oil
[0530] The compound of the invention is dispersed in the lecithin
and arachis oil and filled into soft gelatin capsules.
BIOLOGY EXAMPLE 1
[0531] Bioavailability Testing in Rats
[0532] The bioavailability of compounds of the invention were
compared to the parent compound H2G and other H2G derivatives in a
rat model. Compounds of the invention and comparative compounds
were administered, per oral (by catheter into the stomach), to
multiples of three individually weighed animals to give 0.1 mmol/kg
of the dissolved prodrug in an aqueous (Example 4, 5, Comparative
example 1-3, 5, 8), peanut oil (Comparative examples 4, 9, 10) or
propylene glycol (Example 1-3, 6-12, 17, Comparative example 6, 7)
vehicle dependent on the solubility of the test compound
ingredient. The animals were fasted from 5 hours before to
approximately 17 hours after administration and were maintained in
metabolic cages. Urine was collected for the 24 hours following
administration and frozen until analysis. H2G was analysed in the
urine using the HPLC/UV assay of Stahle & Oberg, Antimicrob
Agents Chemother. 36 No 2, 339-342 (1992), modified as follows:
samples upon thawing are diluted 1:100 in aq dist H.sub.2O and
filtered through an amicon filter with centrifugation at 3000 rpm
for 10 minutes. Duplicate 30 .mu.l samples are chromatographed on
an HPLC column; Zorbax SB-C18; 75.times.4.6 mm; 3.5 micron; Mobile
phase 0.05M NH.sub.4PO.sub.4, 3-4% methanol, pH 3.3-3.5; 0.5
ml/min; 254 nm, retention time for H2G at MeOH 4% and pH 3.33,
.about.12.5 min. Bioavailability is calculated as the measured H2G
recovery from each animal averaged over at least three animals and
expressed as a percentage of the averaged 24 hour urinary H2G
recovery from a group of 4 individually weighed rats respectively
injected i.v. jugularis with 0.1 mmol/kg H2G in a Ringer's buffer
vehicle and analysed as above.
[0533] Comparative example 1 (H2G) was from the same batch as used
for preparation of Examples 1 to 12. The preparation of Comparative
example 2 (monoVal-H2G) and 3 (diVal-H2G) are shown in Examples 20
and 23. Comparative example 4 (distearoyl H2G) was prepared by
di-esterification of unprotected H2G in comparable esterification
conditions to step 2 of Example 1. Comparative examples 5 & 8
(Val/Ac H2G) were prepared analogously to Example 4 using acetic
anhydride with relevant monovaline H2G. Comparative example 6
(Ala/stearoyl H2G) was prepared analogously to Example 6 using
N-t-Boc-L-alanine in step 4. Comparative example 7 (Gly/decanoyl)
was prepared analogously to Example 5 but using the step a)
intermediate made with N-t-Boc-L-glycine. The preparation of
Comparative examples 9 and 10 is shown in Examples 24 and 25
respectively. The results appear on Table 2 below:
3TABLE 2 Compound R.sub.1 R.sub.2 Bioavailability Comparative
example 1 hydrogen hydrogen 8% Comparative example 2 valyl hydrogen
29% Comparative example 3 valyl valyl 36% Example 1 valyl stearoyl
56% Comparative example 4 stearoyl stearoyl 1% Example 2 valyl
myristoyl 57% Example 3 valyl oleoyl 51% Example 4 valyl butyryl
45% Comparative example 5 valyl acetyl 11% Example 5 valyl decanoyl
48% Example 6 valyl docosanoyl 48% Example 7 isoleucyl stearoyl 53%
Example 8 isoleucyl decanoyl 57% Example 9 isoleucyl myristoyl 49%
Example 10 valyl 4-acetylbutyryl 52% Example 11 valyl dodecanoyl
46% Example 12 valyl palmitoyl 58% Example 17 stearoyl valyl 52%
Comparative example 6 alanyl stearoyl 23% Comparative example 7
glycyl decanoyl 25% Comparative Example 8 acetyl valyl 7%
Comparative Example 9 hydrogen stearoyl 12% Comparative Example 10
stearoyl hydrogen 7%
[0534] Comparison of the bioavailabilities of the compounds of the
invention with the comparative examples indicates that the
particular combination of the fatty acids at R.sub.1/R.sub.2 with
the amino acids at R.sub.1/R.sub.2 produces bioavailabilities
significantly greater than the corresponding diamino acid ester or
difatty acid ester. For example, in this model, the compound of
Example 1 displays 55% better bioavailability than the
corresponding divaline ester of Comparative example 3. The compound
of Example 4 displays 25% better availability than the
corresponding divaline ester.
[0535] It is also apparent, for instance from Comparative examples
5, 6 and 7 that only the specified fatty acids of this invention in
combination with the specified amino acids produce these dramatic
and unexpected increases in pharmacokinetic parameters.
BIOLOGY EXAMPLE 2
[0536] Plasma Concentrations in Rats
[0537] A plasma concentration assay was done in male Sprague Dawley
derived rats. The animals were fasted overnight prior to dosing but
were permitted free access to water. Each of the compounds
evaluated was prepared as a solution/suspension in propylene glycol
at a concentration corresponding to 10 mg H2G/ml and shaken at room
temperature for eight hours. Groups of rats (at least 4 rats in
each group) received a 10 mg/kg (1 ml/kg) oral dose of each of the
compounds; the dose was administered by gavage. At selected time
points after dosing (0.25, 0.5, 1, 1.5, 2, 4, 6, 9, 12, 15, and 24
hours after dosing), heparinized blood samples (0.4 ml/sample) were
obtained from a tail vein of each animal. The blood samples were
immediately chilled in an ice bath. Within two hours of collection,
the plasma was separated from the red cells by centrifugation and
frozen till analysis. The components of interest were separated
from the plasma proteins using acetonitrile precipitation.
Following lyophilisation, and reconstitution, the plasma
concentrations were determined by reverse phase HPLC with
fluorescence detection. The oral uptake of H2G and other test
compounds was determined by comparison of the H2G area under the
curve derived from the oral dose compared to that obtained from a
10 mg/kg intravenous dose of H2G, administered to a separate group
of rats. The results are depicted in Table 1B above.
BIOLOGY EXAMPLE 3
[0538] Bioavailability in Monkeys.
[0539] The compounds of Example 1 and Comparative example 3 (see
Biology Example 1 above) were administered p.o. by gavage to
cynomolgus monkeys.
[0540] The solutions comprised:
Example 1
[0541] 150 mg dissolved in 6.0 ml propylene glycol, corresponding
to 25 mg/kg or 0.0295 mmol/kg.
Comparative Example 3
[0542] 164 mg dissolved in 7.0 ml water, corresponding to 23.4
mg/kg or 0.0295 mmol/kg.
[0543] Blood samples were taken at 30 min, 1, 2, 3, 4, 6, 10 and 24
hours. Plasma was separated by centrifugation at 2500 rpm and the
samples were inactivated at 54.degree. C. for 20 minutes before
being frozen pending analysis. Plasma H2G levels were monitored by
the HPLC/UV assay of Example 30 above.
[0544] FIG. 1 depicts the plasma H2G recovery as a function of
time. Although it is not possible to draw statistically significant
conclusions from single animal trials, it appears that the animal
receiving the compound of the invention experienced a somewhat more
rapid and somewhat greater exposure to H2G than the animal which
received an alternative prodrug of H2G.
BIOLOGY EXAMPLE 4
[0545] Antiviral Activity
[0546] Herpes simplex virus-1 (HSV-1)-infected mouse serves as an
animal model to determine the efficacy of antiviral agents in vivo.
Mice inoculated intraperitoneally with HSV-1 at 1000 times the
LD.sub.50 were administered either with a formulation comprising
the currently marketed anti-herpes agent acyclovir (21 and 83 mg/kg
in a 2% propylene glycol in sterile water vehicle, three times
daily, p.o.) or the compound of Example 29 (21 and 83 mg/kg in a 2%
propylene glycol in sterile water vehicle, three times daily, p.o.)
for 5 consecutive days beginning 5 hours after inoculation. The
animals were assessed daily for deaths. The results are displayed
in FIG. 2 which charts the survival rate against time. In the
legend, the compound of the invention is denoted Ex.29 and
acyclovir is denoted ACV. The percentage of mice surviving the
HSV-1 infection was significantly greater following a given dose of
the compound of the invention relative to an equivalent dose of
acyclovir.
[0547] The foregoing is merely illustrative of the invention and is
not intended to limit the invention to the disclosures made herein.
Variations and changes which are obvious to one skilled in the art
are intended to be within the scope and nature of the invention as
defined in the appended claims.
* * * * *